ESGCT 2008 Poster Presentations - Mary Ann Liebert, Inc.

HUMAN GENE THERAPY 19:1098–1202 (October 2008) © Mary Ann Liebert, Inc. DOI: 10.1089/hum.2008.1034

ESGCT 2008 Poster Presentations Taiwan; 2National Cheng Kung University Medical College, Internal Medicine, 1, Dashiue Road, Tainan, Taiwan; 3Medical University of South Carolina, Biochemistry and Molecular Biology, Charleston, United States; 4National Cheng Kung University Medical College, Biochemistry and Molecular Biology, 1, Dashiue Road, Tainan, Taiwan

P1 Enhancing the immune response against melanoma by delivering a plasmid encoding IL-15 to B16.F10 tumor cells via electroporation Bernadette Marrero 1; Kenneth Ugen 1; Richard Heller

2

1University

of South Florida, Molecular Medicine, 12901 Bruce B. Downs Blvd. MDC 7, Tampa, United States; 2Old Dominion University, Bioelectrics, University Suite 5100 830 Southampton Avenue, Norfolk, United States In this study, we are evaluating the effectiveness of an electrically mediated delivery system to induce an anti-tumor response against melanoma. A plasmid encoding the cytokine IL-15 (pIL-15) was delivered directly into tumor cells by electroporation (EP). To enhance the effectiveness of this approach it is necessary to evaluate the criteria for delivery of plasmid DNA using EP conditions, dose, time points and number of treatments that induce sufficient expression to stimulate an appropriate immune response. The evaluation is performed utilizing a dual approach. A mouse melanoma model (B16.F10) was used to study the immune response against melanoma and develop a treatment regimen against established tumors. In addition, we have developed an in vitro 3-D model containing keratinocytes and B16 melanoma cells to evaluate expression levels. The keratinocytes act as a scaffold where melanoma cells can aggregate and expand as though in normal skin. EP parameters from the 3D model were utilized to deliver plasmid IL-15 into tumors. IL-15 protein can elicit an immune response against melanoma antigens by up-regulating CD8 T cells and NK cell response. The expression levels of IL-15 from tumor cells were measured using ELISA techniques obtaining levels of approximately 8-16 fold increase measured at 12hrs and 24hrs after treatment compared to controls. Regression studies were used to evaluate the effectiveness of the treatment parameters in vivo and to further verify that this therapy cures mice and leaves the animals disease-free. Mice were monitored for 105 days post treatments and 62.5% of the mice survived tumor free compared to controls. The goal is to develop a therapy that can be used to stimulate local and systemic immune responses against cancer cells. IL-15 is an excellent candidate for anticancer gene therapy because it stimulates an immune response against tumor antigens and promotes memory responses against reoccurring tumorogenesis.

Background: Angiogenesis plays an important role in the development and progression of most tumors, including lung cancer. Kallistatin exerts anti-angiogenic and anti-inflammatory activities, which may be effective in inhibiting metastasis of lung cancer. The aim of this study was to investigate whether intravenous injection of lentiviral vector encoding kallistatin could suppress experimental metastasis of syngeneic Lewis lung carcinoma (LL2) in mice. Methods: Lentiviral vector encoding kallistatin (LVKallistatin) was constructed, and the expression and bioactivity of kallistatin were verified by ELISA, migration, and invasion assays. C57BL/6 mice were injected with LL2 cells via tail vein on day 0 followed by intravenous injection of LV-Kallistatin, LV-GFP, or saline for 5 days, and the tumor burden and survival time of the mice were monitored. Microvessel density and macrophage infiltration, as well as levels of tumor necrosis factor (TNF)-, transforming growth factor (TGF)-, and kallistatin in the tumors were examined by immunohistochemistry and ELISA. Results: Not only did the conditioned medium from LVKallistatin-treated cells inhibit the migration and proliferation of endothelial cells, it also suppressed migration, invasion, and adhesion of LL2 lung cancer cells. In the experimental lung metastatic model, mice receiving LVKallistatin had lower tumor burden in the lung and survived longer than those receiving LV-GFP or PBS. Microvessel density, macrophage infiltration, and TNF- level in the tumors were decreased, whereas TGF- was slighted elevated 25 days after tumor inoculation. Notably, high level of kallistatin was still detectable in the tumors. Conclusion: Our results demonstrate for the first time that systemic administration of lentiviral vector encoding kallistatin inhibited the growth of metastatic tumor and prolonged the survival of tumor-bearing mice. These findings also suggest that LV-Kallistatin represents a potentially applicable anticancer agent for the treatment of primary and metastatic tumors. E-mail: [email protected] P3

E-mail: [email protected]

Tumour cell–dendritic cell hybrid generation via a fusogenic cell line

P2 Lentivirus-mediated kallistatin gene transfer for the treatment of metastatic lung cancer Ai-Li Shiau 1; Min-Li Teo 1; Che-Hsin Lee 1; Chrong-Reen Wang 2; Julie Chao 3; Lee Chao 3; Chao-Liang Wu 4 1National Cheng Kung University Medical College, Microbiology and Immunology, 1, Dashiue Road, Tainan,

Isabelle Blangenois 1; Siew Chiat Cheong 2; Jean-Denis Franssen 3; Thierry Velu 1; Catherine Yourassowsky 4; Frank Dubois 4; Annick Brandenburger 1 1Université

Libre de Bruxelles, IBMM-IRIBHM, Gosselies, Belgium; 2Université Libre de Bruxelles, IBMM-IRIBHM, Belgium; 3Euroscreen, *, Gosselies, Belgium; 4Université Libre de Bruxelles, MRC, bld Roosevelt, Bruxelles, Belgium

1098

ESGCT 2008 POSTER PRESENTATIONS Hybrids obtained by fusion between tumour cells and dendritic cells (DC) have been proposed as anti-tumour vaccines because of their potential to combine the expression of tumour-associated antigens with efficient antigen presentation. The classical methods used for fusion, polyethylene glycol and electrofusion, are cytotoxic and generate cell debris that can be taken up by dendritic cells rendering the identification of hybrids difficult. Another approach to cell fusion uses the expression of viral fusogenic glycoproteins (FMG) via the transduction of one of the partners. However; the transduction step can be rate limiting for certain cell types and it requires additional manipulation of cells. We have therefore developed a method to circumvent the need for transduction by establishing a cell line that stably expresses the Gibbon Ape Leukaemia Virus - FMG. This cell line is not itself susceptible to fusion and it has been used to generate hybrids between tumour cells and dendritic cells. This approach further offers the possibility to modulate the immune response by the addition of appropriate genes to the cellular fusion agent. Determining the kinetics of fusion is an important parameter to define the time point at which an optimal number of small hybrids (ideally 1 cell of each fusion partner) is generated. Small hybrids should be more stable and able to migrate to lymph nodes where they activate cytotoxic T lymphocytes. A digital holographic microscope developed at the ULB (MRC) allows to monitor fusion events over time by measuring the local cell thickness with a few nanometers accuracy. The instrument provides also a refocusing capability of cells located at different depths. Therefore, the fusion analysis can be started right after mixing of cells, before they settle to the bottom of the culture dish. This property is important for our approach i.e. the fusion of non-adherent DCs with adherent CHO-FMG and tumour cells. With this approach we could follow the generation of hybrids between cells either expressing eGFP or labelled with Qtracker650 nanoparticles. The two types of fluorescence can be distinguished by the pattern of labelling (uniform distribution in the cytoplasm for eGFP or very bright spots for Q-dots). E-mail: [email protected] P4 Efficient immunotherapy for liver cancer with a synthetic vaccine in a transgenic mouse model with Galactosidase as target tumour antigen Jeannette Cany 1; Dorian McIlroy 2; Nicolas Ferry 1; Bruno Pitard 3; Sophie Conchon 1 1EA4274, INSERM CIC4, Biotherapies Hepatiques, Nantes, France; 2CHU Hotel Dieu, JE 2437, Laboratoire de Virologie, Nantes, France; 3INSERM U915, Institut du Thorax, In-CellArt, Nantes, France

Background: To optimize vaccination strategies for immunotherapy in the liver, we have generated a line of transgenic mice expressing -Galactosidase (bGal) downstream of the alpha-fetoprotein promoter (AFP/Gal). Methods: Gal expression was documented by qRT-PCR, enzyme activity and immunohistochemistry. Gal-specific CD8T-cell activation in mice immunised with various vectors was measured by interferon- ELISpot. Results: Like AFP, Gal expression was detected in fetal hepatocytes and disappeared around birth. In adult mice, a CD8 T-cell response to Gal was observed after immunisation with Gal adenovirus or plasmid DNA but not with

1099 Gal protein or after retroviral infection. When Gal was reexpressed in normal adult hepatocytes, immunisation with Gal adenovirus triggered T-cell mediated elimination of Gal-expressing hepatocytes. However the response was weaker than in AFP/Gal animals in which bGal was only present around birth. AFP/Gal mice were then vaccinated with Gal-expressing vectors (adenovirus, DNApolymer) followed by tumour challenge consisting of a sub-cutaneous injection of Hepa1.6 cells stably expressing Gal. Tumour size was monitored for 25 days following tumour challenge. In the control group 4 out of 8 animals had a tumour of 250mm3 or more whereas 8 out of 8 of the animals vaccinated with the synthetic DNA  polymer formula controlled the tumour growth, with a complete regression in 5 of these mice (p  0.05 vs control). Although IFN ELISpot revealed a strong Gal-specific cytotoxic response in all vaccinated animals, no protective effect of the adenoviral vaccination was observed in this experiment. Conclusion: In AFP/Gal mice Gal is a fetal liver selfantigen. Adenoviral immunisation allows the complete elimination of Gal-expressing normal hepatocytes in adult animals. However in a sub-cutaneous tumour challenge model this vaccine is not protective. A DNApolymer formula brings a significant protection in the same model. These vaccines are now being tested in intra-hepatic tumour growth models and against real tumour antigens. Our results validate the AFP/Gal mice as an interesting background to test immunotherapy strategies in hepatocarcinogenesis models. E-mail: [email protected] P5 Treatment-induced S-phase checkpoint activation is associated to sensitivity to TK/GCV Daniel Abate-Daga 1; Cristina Fillat

2

1Centre

de Regulació Genòmica-PRBB, Programa Gens i Malaltia, Barcelona, Spain; 2Centre de Regulació GenòmicaPRBB., Programa Gens i Malaltia, CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain The Herpes simplex virus Thymidine Kinase (TK)/Ganciclovir (GCV) suicide system has been extensively assayed as a gene therapy approach to treat cancer. Although a plethora of in vitro studies, as well as preclinical and clinical trials, have shown a great potential for TK/GCV to have an impact in clinical practise, little is known about its mechanism of action. Several authors have reported that S-phase and/or G2/M-phase arrest and subsequent apoptotic cell death are triggered in response to treatment. On this basis, we aimed to identify the molecular effectors governing these cell cycle alterations and to evaluate the relevance of these cellular events preceding apoptosis as a part of the mechanism of action of TK/GCV. We first characterized the sensitivity of a panel of p53-deficient pancreatic cancer cell lines to TK/GCV by estimating the Inhibitory Dose of 50% (ID50) of an adenoviral vector carrying the TK cDNA (AdTK). This parameter was used to classify tumor cells as sensitive (NP-18, BxPC-3, RWP-1) or resistant (NP-9, NP-31, MIAPaca-2, PANC-1) to TK/GCV. When treated at isotoxic viral doses (ID50), sensitive lines displayed a marked increase in S-phase or S- and G2/Mphase cells. However, resistant lines showed milder or even null alterations in cell cycle profiles. In sensitive NP-18 cells, S-phase delay was concomitant with a decrease in DNA synthesis. Moreover, phosphorylation of histone H2AX, Chk1 and Chk2 was observed in response to treatment, before in-

1100 duction of apoptosis.Treatment with UCN-01, an inhibitor of Chk1 activity, induced an antagonistic effect on TK/GCVinduced cell cycle arrest, DNA synthesis inhibition and cytotoxicity. Altogether, our results suggest that cell cycle alterations in response to TK/GCV treatment may play an important role in its mechanism of action and might have a predictive value in terms of treatment output. E-mail: [email protected] P6 In vivo interactions of systemically delivered adenovirus serotype 5 in mice Anniina Koski 1; Maria Rajecki 1; Kilian Guse 1; Anna Kanerva 2; Sari Pesonen 2; Akseli Hemminki 2 1University

of Helsinki & HUCH & FIMM, Molecular Cancer Biology Program &, Transplantation laboratory, Helsinki, Finland; 2University of Helsinki & HUCH & FIMM, Molecular Cancer Biology Program &, Department of Obstetrics and Gynecology, Helsinki, Finland Background: The successful systemic use of adenoviruses for cancer gene therapy is hindered by unfavorable bioavailability. Intravenously delivered adenoviruses are rapidly cleared from blood by macrophage lineage cells of the liver and spleen and have also been reported to bind to platelets. Vitamin K –dependent coagulation factors, factor IX and X in particular, have been suggested as key mediators for the adenovirus liver tropism. The aim of this study is to determine the effects of coagulation factor, thrombocyte and liver macrophage (Kupffer cell) ablation on biodistribution of serotype 5, Ad5luc1, and capsid modified adenoviruses, Ad5/3luc1 and Ad5pK7(GL), in mice with orthotopic breast tumors. Methods: Prior to intravenous injection of adenoviruses (Ad), mice with orthotopic breast tumors were pretreated with warfarin–an inhibitor of vitamin K dependent coagulation factor synthesis, GPIIb-antibody–an anti-platelet antibody that causes thrombocytopenia or poly(i)–an inhibitor of the Kuppfer cell scavenger receptor, or a combination of these. Virus availability in blood 10 min after injection was determined from blood samples. 72 hours afterwards mice were given luciferine intraperitoneally and imaged with an In Vivo Imaging System for luciferase expression. Tumors and tissues were then collected and analysed by luciferase assays. Results: Coagulation factor ablation with warfarin pretreatment resulted in reduction of gene delivery to liver, spleen and lung. Kupffer cell depletion by poly(i) resulted in increased persistence of Ad in blood but did not affect biodistribution significantly. Depletion of Kupffer cells in combination with thrombocytopenia, induced by GPIIb antibody, doubled the systemic gene delivery of 5/3 chimeric Ad to orthotopic breast tumors (p  0.05). Finally, the triple ablation of platelets, Kupffer cells and coagulation factors increased the tumor to liver ratio of systemic Ad gene delivery by 81% (p  0.05). Conclusion: Depletion of coagulation factors can reduce transduction of liver, spleen and lung. Kupffer cell depletion is the most feasible method of increasing amount adenovirus in systemic blood flow and in combination with ablation of thrombocytes can increase the transduction of adenovirus to tumors. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P7 Isolation of a mutant reovirus T3D with improved oncolytic activity in malignant glioma Sanne van den Hengel 1; Diana van den Wollenberg 2; Martine Lamfers 3; Peter Sillevis Smitt 4; Rob Hoeben 2 1Erasmus University Medical Center, Neuro Oncology, Rotterdam, Netherlands; 2Leiden University Medical Center, Molecular Cell Biology, Leiden, Netherlands; 3Erasmus University Medical Center, Neurosurgery, Rotterdam, Netherlands; 4Erasmus University Medical Center, Neurology, Rotterdam, Netherlands

Malignant gliomas are the most common type of primary brain tumors. Median survival with current treatments (chemotherapy, radiation and surgical removal) is less then a year. The search for new alternative treatments is ongoing and the use of viruses as oncolytic agent is one of the research fields. A virus of interest is the human Orthoreovirus Type 3 Dearing (T3D). It is a non-enveloped double stranded RNA virus containing ten genome segments. Although this virus is commonly isolated of the gastrointestinal and respiratory track no disease symptoms are linked to it in humans. Previous studies have demonstrated oncolytic activity of this virus against several cancers. So far, no maximum tolerated dose was reached in thirteen human clinical I trials involving administration of reovirus T3D. Tumour mass is a heterogeneous population of cells, low expression of the high affinity receptor limits the oncolytic activity. The high affinity receptor for reovirus T3D is the Junction Adhesion Molucule-A (JAM-A). Modification of the viral capsid proteins may be used to increase its activity in JAM-A negative cells. A T3D mutant with increased activity was isolated after selection on the JAM-A negative malignant glioma cell line U118MG. This highly effective virus was tested on several malignant glioma cell lines and primary malignant glioma isolates. A clear cytopathic effect was observed. Infection was further confirmed by immunofluorescence staining with an anti-dsRNA antibody. The mutant Reovirus T3D variant, but not wild-type T3D, efficiently infected cultured cells from freshly isolated gliomas and exhibited signs of lytic activity and accumulation of dsRNA in the cytoplasm. Our data suggest that this mutant is an interesting new candidate for use as oncolytic agent in the treatment of malignant glioma. E-mail: [email protected] P8 Heat and rapamycin-based gene switches for spatial and temporal control of transgenes Francisco Manuel Martin-Saavedra 1; Alba Bore 1; Richard Voellmy 2; Nuria Vilaboa 1 1Hospital

Universitario La Paz-CIBER BBN, Unidad de Investigación, Madrid, Spain; 2HSF Pharmaceuticals S.A, HSF Pharmaceuticals S.A, Pully, Switzerland The development of transcriptional targeting strategies to ensure tight control over the expression of therapeutic proteins remains an important goal in current gene therapy. We have previously developed several gene switches that can provide for both spatial and temporal regulation of transgene activity. These switches utilize the strictly heat-regulated human hsp70B promoter to trigger expression of a small molecule-activated transactivator that maintains its own expression by an autoregulatory loop and transactivates

ESGCT 2008 POSTER PRESENTATIONS a target gene in the presence of the appropriate small molecule ligand. We report here on a new version of the heattriggered, small-molecule-activated gene switch which employs a dimerizer-controlled chimeric transactivator composed by two modules derived from the human proteins FKB12 and FRAP fused to a DNA binding domain and a transcriptional activation domain, respectively. We created a reversible switch capable of mediating sustained gene activity subsequent to activation by transient heat in the presence of rapamycin or its non-immunosuppressive analog AP21967. Luciferase reporter gene expression was subjected to the control of the switch, which was tested in human tumor cell lines. Heat-activation of the system in the presence of rapamycin or AP21967 increased luciferase activity by several orders of magnitude, while the reporter activity induced by heat treatment or dimerizer alone was negligible. The ability of the heat-activated and rapalog-dependent gene switch to control the expression of HSV thymidine kinase gene was tested in transient transfection experiments. Heat-activation of the system in the presence of rapamicyn highly decreased cell viability upon supply of the prodrug ganciclovir. E-mail: [email protected] P9 Nanoliposomal delivery of Par-4 to colon tumor cells Mark Kester 1; Christina Kline 2; Danielle Pastor 3; Norman Lee 4; Rosalyn Irby 2 1Pennsylvania

State University College of Medicine, Pharmacology, Hershey, United States; 2Pennsylvania State University College of Medicine, Medicine, Hershey, United States; 3Pennsylvania State University College of Medicine, Surgery, Hershey, United States; 4George Washington University Medical Center, Pharmacology, Washington, DC, United States Background: An estimated 400,000 people die of colorectal cancer yearly worldwide. Often colon cancer related mortality results from metastases that are present at the time of diagnosis. Despite a combination of 5-fluorouracil (5-FU) with other chemotherapeutic agents, the clinical response rate for patients with metastatic colon cancer remains 2039%, indicating a need for a more effective regimen. The tumor suppressor, prostate apoptosis response protein 4 (Par4), has been shown to have diminished expression in a number of cancers, particularly those refractory to chemotherapy. Increased Par-4 can sensitize cells to chemotherapy through inhibition of NFB. Nanoliposomes offer a means of delivering genes to cells in vivo to confer enhanced sensitivity to chemotherapy. Methods: Par-4 effects: Human colon cancer cells were examined for Par-4 expression levels, activity, and downstream effects. The cells were transfected to overexpress Par-4 to observe the effects of increased Par-4 on sensitivity to 5-FU. Downstream effects of Par-4 were examined using a gene expression array. Nanoliposomal delivery of Par-4 plasmid: Cells were incubated with cationic nanoliposomes loaded with Par-4 plasmid (1 g/ml). After 48 hours, the cells assessed for Par-4 expression. Results: Overexpressed Par-4 increased apoptosis in the presence of 5-FU. Western blotting confirmed Par-4 levels and the association of Par-4 with NFB. Microarray results supported previous findings that Par-4 inhibits NFB activity in that many of the proteins shown to be downregulated by Par-4 have NFB binding sites in the promoter region.

1101 Nanoliposome delivery of Par-4 plasmid to cells was both effective and non-toxic, compared to traditional transfection agents in this study. Conclusion: Nanoliposomes provide an effective, nontoxic method of delivering genes to tumor cells. The tumor suppressor Par-4 is a promising candidate for use as gene therapy, and the nanoliposomal delivery system makes it a viable therapy for in vivo targeting of tumor cells, offering an effective treatment regimen for metastatic colon cancer. E-mail: [email protected] P 10 Immunotherapy for malignant glioma using lentivirally transduced dendritic cells in an experimental murine model Wim Maes 1; Jaan Toelen 2; Marianne Carlon 2; Rik Gijsbers 2; Zeger Debyser 2; Jan Cuppens 1; Stefaan Van Gool 1 1KU Leuven, Clinical Immunology, Leuven, Belgium; 2KU Leuven, Molecular and Cellular Medicine, Laboratory for Molecular Virology and Gene Therapy, Leuven, Belgium

Background: Effective induction of systemic cellular antitumor immunity is crucial for immunotherapeutic gene therapy against intracerebral gliomas. We examined in this study the in vivo efficacy of prophylactic subcutaneous (s.c.) immunization with dendritic cells (DC) transduced with lentiviral vectors (LV) in the GL261 orthotopic glioma model. Moreover, immune monitoring on brain infiltrating lymphocytes was performed. Methods: VSV-G pseudotyped LV encoding eGFP driven by a CMV promoter (LV-eGFP) were produced in 293T cells under serum free conditions. Bone-marrow derived mouse DC were transduced ex vivo with these LV and matured with LPS. C57BL/6 mice were vaccinated with a s.c. injection of 1  106 DC and were subsequently stereotactically injected with GL261 glioma cells expressing eGFP. Control mice were left untreated. In both groups mice were sacrificed at day 5 (early) and day 30 (late) after tumor challenge: time points and tumor infiltrating lymphocytes were analysed by flowcytometry. Animals were clinically monitored for tumor-induced neurologic deficit and mortality was compared between the two groups. Results and Conclusion: Although not protective, survival of DC treated mice was significantly prolonged compared to untreated animals (Logrank p  0.001). Both early and late immune monitoring revealed a significant increase in the absolute number of brain infiltrating lymphocytes. Moreover, total CD4 and CD8 as well as activated (CD44) CD4 and CD8 T cells were significantly increased by DC treatment. These data underscore the feasibility of glioma immunotherapy based on ex vivo transduction of DC with lentiviral vectors. E-mail: [email protected] P 11 Killing activity of suicide gene therapy against cancer stem cells Keiji Shimizu 1; Kazuhiro Ikenaka 2; Yu Kwawnishi 1; Masakazu Tamura 1; Toshio Yawata 1 1Kochi Medical School, Department of Neurosurgery, Nankoku, Japan; 2National Institute for Physiological Sciences, Division of Neurobiology and Bioinformatics, Okazaki, Japan

1102 Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells. Both transduction efficiency and gap junction mediated bystander effect are essential factor for the anti-tumor effect by the HSV-TK/GCV suicide gene therapy. We established the packaging cell line PAMP51 producing high-titer retrovirus expressing suicide gene HSV-TK and concentrated the virus with titers as high as 1-1011-12 c.f.u. /ml by low speed centrifugation. Using this retroviral vector, mouse glioma model was completely cured by administration of ganciclovir (GCV). Recently, several investigators identified a small population of cancer stem cells in brain tumor tissues and cell lines. These cancer stem cells form spheres and maintain self-renewal capacity, tumorigenecity and multiple drug resistance. The existence of the cancer stem cells is thought to be a cause for recurrence of the tumors after treatment with chemotherapy, radiotherapy and surgical resection. Therefore, we investigated the therapeutic efficacy of suicide gene therapy for the cancer stem cells derived from glioma cells. The cancer stem cells isolated from glioma cells by sphere culture preferentially expressed stem cell marker, CD133. The growth rates of the cancer stem cells were lower than parental cells. A low transduction rate by retrovirus vectors was observed in cancer stem cells, but not in parent cells. Because the cancer stem cells expressed retrovirus receptors as well as parent cells, the transduction efficiency of cancer stem cells is thought to be growth rate dependent. But importantly, cancer stem cells expressed connexin43, a component of gap junction. This result suggests that non-transduced cancer stem cells can be killed via bystander effect. E-mail: [email protected] P 12 Transductional retargeting of adenovirus-5 to alphavbeta6 integrin Lynda Coughlan 1; Iain McNeish 2; Georges Vassaux 3; John Marshall 1; Ian R. Hart 4; Gareth Thomas 4 1Institute of Cancer, Dept. Tumour Biology, Bart’s and the London School of Medicine, London, United Kingdom; 2Institute of Cancer, Dept. Molecular Oncology, Bart’s and the London School of Medicine, London, United Kingdom; 3INSERM, CIC-04, EA 4274 Biothérapies Hépatiques, and Institut des Maladies de l’Appareil Digestif, CHU Hôtel Dieu, Nantes, Nantes, France; 4Institute of Cancer, Dept. Tumour Biology, Bart’s and the London School of Medicine, London, United Kingdom

Background: v6 integrin is not expressed constitutively on normal tissue but is significantly upregulated in many carcinomas, including head and neck squamous cell carcinoma (HNSCC), pancreatic, stomach, ovarian and in lung and colorectal carcinomas where it has been identified as a prognostic indicator. We have previously identified and characterised a high affinity v6-selective peptide, A20FMDV2 and in this study report the genetic incorporation of this targeting ligand into the HI loop of Ad5, in an attempt to redirect the tropism to v6 integrin. Methods: Preliminary experiments were carried out using A20-modified recombinant Ad5 knob protein. The ability of KnobA20 to bind to v6 on BT-20 cells, and to competitively inhibit binding of an v6-specific antibody, was assessed by flow cytometry. KnobA20 inhibition of v6-dependent function in cell migration was assessed on high

ESGCT 2008 POSTER PRESENTATIONS v6-expressing V6 cells. An A20 fiber modified virus, Ad5-EGFPA20, was constructed featuring an EGFP transgene within the E3 region. A panel of eight v6-positive carcinoma cell lines with differential expression of adenovirus receptors, CAR and v3/5, were used to compare the infectivity and cytotoxicity of Ad5-EGFPA20 in parallel with its control, Ad5-EGFPWT. Results: KnobA20 inhibited v6-dependent cell migration in a manner comparable to an v6-specific antibody. The successful attachment of KnobA20 and its subsequent inhibition of both v6-specific antibody binding, and v6mediated infection with Ad5-EGFPA20, was determined by flow cytometry. KnobA20 inhibition was dose dependent [IC50 0.008g protein/10(5) cells; IC50 0.03g protein/10(5) cells, respectively] whereas KnobWT did not demonstrate an effect. Ad5-EGFPA20 exhibited up to 50-fold increases in CAR-independent transduction on a panel of v6-positive human carcinoma lines (MOI 10). The infectivity enhancement observed also corresponded with up to 480-fold increased cytotoxicity in cells infected with Ad5-EGFPA20 compared with Ad5-EGFPWT. Conclusion: These data highlight the potential of this construct as a prototype vector for improved delivery to v6positive tumours in vivo. E-mail: [email protected] P 13 Increased relapse-free survival time for fibrosarcoma bearing cats after adjuvant intratumoral magnetofection applying different cytokine genes Ulrike Schillinger 1; Anika Jahnke 2; Cornelia Fischer 2; Florian Walsch 2; Bianca Schwarz 2; Tina Kempf 2; Johannes Hirschberger 2; Roberto Köstlin 2; Thomas Brill 1; Bernd Gänsbacher 1 1Technische

Universität München, Institute of Experimental Oncology, Munich, Germany; 2Ludwig-MaximiliansUniversität, Department of Small Animal Surgery, Veterinary School, Munich, Germany Feline fibrosarcoma is an everyday challenge in veterinary practice. Despite aggressive pre- or post-operative treatment it has a high relapse rate of aprox. 75 % within 6 months after surgical resection. This makes it an ideal model for evaluating immunostimulatory therapeutic strategies. Here we report preliminary results from a comparative clinical study where the genes for feline GM-CSF, IFN-g and IL-2 in combination, feline GM-CSF alone or human GM-CSF were administered. The study design is prospective, randomized, placebo-controlled ( standard therapy) and includes four arms: (1) standard therapy, i.e. surgery alone; (2) nonviral Magnetofection of the feline GM-CSF, IFN-g and IL-2 genes into the tumor before surgery or nonviral administration by Magnetofection of feline (3) or human GMCSF (4) gene alone. The procedure included phase I dose finding studies for the gene therapy groups followed by a phase II. Preliminary clinical endpoint is relapse-free survival for one year. Magnetofection, which has been developed in our laboratory, is the association of vectors with magnetic particles (chemicell, Berlin, Germany) and gene delivery under the influence of a magnetic field. It was applied here in order to achieve improved retention of the injected vector dose in the tumor. Pre- and postsurgical diagnosis included complete clinical monitoring of the cats. All gene-therapeutic treatments were well tolerated and led to significantly prolonged re-

ESGCT 2008 POSTER PRESENTATIONS

1103

lapse-free survival. This is encouraging with respect to future use in veterinary practice as this treatment can be easily administered. With respect to potential human clinical use, the results are promising as they have been obtained with a naturally grown tumor in real patients and not in a tumor model with inbred mice. Support by the German Federal Ministry of Education and Research, grant 13N9064 is gratefully acknowledged. E-mail: [email protected] P 14 Adenovirus-mediated systemic therapy of medullary carcinoma in the thyroid glands of RETC634R transgenic mice through targeted expression of RETTK Anke Schmidt 1; Nadine Sommer 1; Leona Trübe 1; Christian Eipel 2; Uta Dahmen 3; Brigitte M. Pützer

1

1University

of Rostock, Biomedical Research Center, Department of Vectorology and Exp. Gene Therapy, Rostock, Germany; 2University of Rostock, Experimental Surgery, Rostock, Germany; 3University Hospital Essen, General, Visceral and Transplantation Surgery, Essen, Germany Background: Efficient and specific killing of neoplastic cells, in particular tumor metastases, remains a major challenge of current cancer therapy modalities. Medullary thyroid carcinoma (MTC) based on activating mutations in the RET proto-oncogene encoding a receptor tyrosine kinase is characterized by aggressive tumor growth and early metastasis, and thus represents an attractive model for gene-based therapeutic strategies. A precondition for successfull eradication of speaded tumor cells are cancer targeted gene delivery systems that can be systemically applied. We have previously identified MTC specific peptides by intravenous injection of a phage display peptide library into RET-C634R transgenic mice carrying orthotopic tumors. Methods: Here, we chemically linked these peptides to genetically modified (CAR depleted) Ad vectors expressing a dominant-negative truncated form of the receptor (RETdeltaTK) and analyzed its anticancer capacity compared to AdRETdeltaTK with native tropism and AdGFP linked to specific ligand after systemic vector administration. Results: Two weeks after virus injection into the tail vein of transgenic mice with growing MTC in their thyroid glands, tumors were strongly reduced in mice treated with specifically-tagged AdRETdeltaTK, whereas the tumor size in mice injected with native AdRETdeltaTK remained stable. In contrast, tumors in mice treated with the AdGFP-peptidevector were larger than their initial size. Quantitative PCR, Western blotting, and immunohistochemistry of mouse tissues revealed direct binding of the peptide-tagged AdRETdeltaTK to MTC. Biodistribution of the vectors was monitored by in vivo-imaging and intravital microscopy, clearly indicating that gene transfer mediated through peptidetagged vectors is redirected to neoplastic cells by reducing binding to normal target cells. Conclusion: This suggests that the corresponding receptor is predominantly expressed on cancerous tissue. Our data indicate that Ad vectors coupled to tumor selective peptides are suitable to improve the efficiency of therapeutic gene transfer to systemic tumor cells, thereby providing the basis for an antimetastatic therapy. Work was supported by DFG grants PU188/5-2, PU188/53 and the FORUN program of Rostock University Medical Faculty. E-mail: [email protected]

P 15 B7-H1 mediates modified adenoviral vector gene delivery into murine leukemic residual cells Elodie Grellier ; Sophie Rogee ; Bruno Quesnel ; JC D’Halluin ; Morvane Colin INSERM, U837, Lille, France Tumor dormancy is characterised by the persistance of residual tumor cells that are able to grow and to spread, but are maintained in an equilibrium state into the organism during several years. Despite the mechanisms implied in the escape to the immune system is yet unknown, it appears that adaptative T-cell immunity maintains dormancy. Eradication of tumor dormant cells is an important goal towards the definitive cure of malignancies. Among the therapeutic hopes resides the possibility to specifically target such residual cancer cells. Targeting could be obtained using adenoviral vectors that are able to infect a wanted cell in order to kill it selectively. We demonstrated that a genetically fiber modified adenoviral vector (HAdV-5-F2/BAdV-4) is able to specifically deliver gene into murin leukemic residual cells (LRCs) and its ability is strongly correlated to the cell surface expression of B7-H1. B7-H1 is a B7-related protein that inhibits T-cells activation in specific cytotoxic T-cells (CTLs). B7-H1 is highly expressed by LRCs as well as in a broad range of human cancer tissue and is implicated in the establishment of tumor dormancy. As LRCs had enhanced B7H1 expression proportional to the time they had persisted in the host, HAdV-5-F2/BAdV-4 represents a good tool to specifically eradicate these cells. The mechanism leading to its entry into LRCs is not clearly established but it appears that B7-H1 could allow viral uptake. B7-H1 is the ligand of receptor programmed death 1 (PD1) that mediate a co-inhibitory signal to the lymphocyte T preventing its activation. The secretion of PD1 by dormant cells would block B7-H1 and restore the activation of T-cells in CTLs, inducing specific dormant tumor cell destruction. We then decided to produce a HAdV-5-F2/BAdV-4 carrying the gene encoding for the soluble part of PD1 to infect dormant cells and selectively kill them. A bioluminescence model of LRCs has been established in our laboratory to monitor eradication of tumor cells after injection of HAdV-5-F2/BAdV-4sPD1. To extend our results to human cells, we also demonstrated that HAdV5-F2/BAdV-4 is able to specifically infect human cells overexpressing B7-H1. It could then also be a good candidate to eradicate human LRCs by targeting B7-H1 at their cell surface. E-mail: [email protected] P 16 A new chimeric enzyme for fluorouridine activation in cancer suicide gene therapy Claudia Minici ; Laura Muzzolini ; Paola Tornaghi ; Massimo Degano San Raffaele Scientific Institute, Biocrystallography Unit, Milan, Italy Transfer into cancer cells of a gene encoding a prodrugactivating enzyme is an attractive method to bypass the low efficiency of endogenous conversion. 5-Fluorouridine (FUR) is a precursor of the antineoplastic agent 5-Fluorouracil (FU) that is more bioavailable and tolerated than FU, but it is inefficiently converted to the active metabolite 5-Fluorouridine monophosphate (FUMP) in vivo.

1104 We engineered a chimeric protein that is able to catalyze in vitro the conversion of FUR into FUMP, termed FAE for fluorouridine-activating enzyme. Different tumor cell lines (HeLa, CaCo-2, Hep G2 and TRAMP-C1) were transfected with a FAE-encoding vector and treated with varying concentrations of FUR. All cell lines expressing FAE showed an increased sensitivity towards FUR, with an EC50 reduction ranging from 66% to 97%. Cells resistant to high FUR doses decreased their survival up to 62% when expressing the chimeric protein. The effects were persistent even in the presence of only 20% of cells transfected, thus suggesting a pronounced bystander effect. This represents a remarkable advantage in suicide gene therapy because it could counterbalance a suboptimal efficiency of in vivo gene transfer. The reduction of FUR EC50 in FAE-expressing cells could allow lower prodrug doses in treatment, thus reducing systemic toxicity, and the decreased number of surviving cells could minimize the possibility of tumor recurrences. These qualities reinforce the therapeutic potential of the FAEFUR system. Our future aim is to verify the effects of the chimeric enzyme in cancerous tissues, and to assess its efficacy and safety in animal models. To this purpose we are developing an adenovirus-mediated transfer system of FAE. E-mail: [email protected] P 17 Pancreatic ductal adenocarcinoma (PDAC) patients reveal elevated TRAIL Death Receptor-4 (DR4) and decoy receptor-2 (DcR2) expression correlated with significant cell death Ahter Dilsad Sanlioglu 1; Ercument Dirice 1; Ozlem Elpek Aylin Fidan Korcum 3; Mustafa Ozdogan 4; Inci Suleymanlar 5; Mustafa Kemal Balci 6; Thomas Griffith 7; Salih Sanlioglu 1 2;

1Akdeniz

University Faculty of Medicine, Human Gene Therapy Unit, Medical Biology and Genetics, Antalya, Turkey; 2Akdeniz University Faculty of Medicine, Pathology, Antalya, Turkey; 3Akdeniz University Faculty of Medicine, Radiation Oncology, Antalya, Turkey; 4Akdeniz University Faculty of Medicine, Division of Oncology, Antalya, Turkey; 5Akdeniz University Faculty of Medicine, Division of Gastroenterology, Antalya, Turkey; 6Akdeniz University Faculty of Medicine, Division of Endocrinology and Metabolism, Antalya, Turkey; 7University of Iowa, College of Medicine, Urology, Iowa City, United States Objectives: The importance of TRAIL and TRAIL receptor expression in pancreatic carcinoma development is not known. To reveal the putative connection of TRAIL and TRAIL receptor expression profile to this process, we analyzed and compared the expression profile of TRAIL and its receptors in pancreatic tissues of both non-cancer patients and patients with pancreatic ductal adenocarcinoma (PDAC). Methods: Thirty one non-cancer patients and thirty-four PDAC patients were included in the study. TRAIL and TRAIL receptor expression profiles were determined by immunohistochemistry. Annexin V binding revealed the apoptotic index in pancreas. Lastly, the tumor grade, tumor stage, tumor diameter, perineural invasion, and number of lymphnode metastasis were used for comparison purposes. Results: TRAIL decoy receptor-2 (DcR2) and death receptor-4 (DR4) expression were up-regulated in PDAC patients

ESGCT 2008 POSTER PRESENTATIONS compared to non-cancer patients, and the ductal cells of PDAC patients displayed significant levels of apoptosis. In addition, acinar cells from PDAC patients had higher DcR2 expression but lower DR4 expression. Increased DcR2 expression was also observed in Langerhans Islets of PDAC patients. Conclusions: Differential alteration of TRAIL and TRAIL receptor expression profiles in PDAC patients suggest that the TRAIL/TRAIL receptor system may play a pivotal role during pancreatic carcinoma development. E-mail: [email protected]

P 18 Silencing of K-ras reduces the proliferation of colon adenocarcinoma cells Suzana Mesojednik ; Bostjan Markelc ; Urska Kamensek ; Gregor Tevz ; Simona Kranjc ; Gregor Sersa ; Maja Cemazar Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia Background: Activation of K-ras oncogene is implicated in colorectal carcinogenesis, where it is mutated in 30-50% of the adenocarcinomas, hence it is a strong candidate for drug development. In the present study, we used siRNAs targeting K-ras to downregulate human Kras expression in colon adenocarcinoma cell lines HT29 and Lovo. Methods: Three different siRNA duplexes based on human K-ras gene sequence were introduced into HT29 and Lovo cells in vitro by RNAiMax Lipofectamin as a vehicle. The effects of siRNA sequences on mRNA K-ras levels were determined by RT-PCR analysis. For stable and cost effective K-ras silencing, the plasmid DNA encoding microRNA of the most efficient siRNA was designed and introduced into Lovo cells by electroporation. Efficiency of electoporation as a delivery method was evaluated with fluorescence microscopy and Western blot analysis using plasmid DNA encoding reporter shRNA directed against green fluorescence protein (GFP), which was introduced into murine LPB cells that stably express GFP. The effects of siRNA molecules on cell proliferation were evaluated by MTS and clonogenic assay. Results: RT-PCR analysis showed that all siRNAs reduced the expression of K-ras mRNA. The most effective were two K-ras siRNAs denoted 53 and 393, reducing K-ras mRNA levels up to 80% compared to negative control. However, only K-ras siRNA 393 reduced cell proliferation (20%) of LoVo and HT29 cells for 6 days, while K-ras siRNA 53 and scrambled siRNA did not affect cell growth. Electroporation proved to be an effective delivery method to introduce plasmid DNA encoding shRNA into cells to silence GFP gene, resulting in 20% silencing. Similarly to siRNA, electrotransfered plasmid DNA encoding K-ras miRNA 393 resulted in 20% reduction of proliferation in Lovo cells. The effect of silencing was still present at day 7. Conclusion: The present study shows that all tested K-ras siRNAs efficiently reduced the expression of K-ras mRNA in HT29 and Lovo cell lines, while the anti-proliferative effect of K-ras siRNA was siRNA sequence specific. K-ras silencing by plasmid DNA encoding K-ras miRNA 393 showed the same efficiency as siRNA duplexes. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 19 rna based immuno-gene therapy of rat glioma Roche Frank 1; Brian Sheahan 2; Shane O Mara 3; Gregory Atkins 1 1Trinity

College Dublin, Microbiology, Dublin, Ireland; College Dublin, Veterinary Pathology, Dublin, Ireland; 3Trinity College Dublin, Trinity College Institute of Neuroscience, Dublin, Ireland 2University

Background: Glioblastoma Multiforme (GBM) is the most common and most malignant brain tumour in humans and current therapeutic interventions rarely extend survival beyond 2 years. The Semliki Forest Virus – Virus Like Particle (SFV-VLP) system is a non-replicating expression system previously used in a variety of murine tumour models. This project is designed to examine the anti-tumor effect of SFV VLPs encoding Interleukin 12 (IL-12), delivered intracerebrally, and Interferon gamma (IFN-) or Interferon beta (IFN-), delivered intranasally, on two rat models of GBM. Methods: In vitro studies examined the expression and activity of cytokine encoding SFV expression vectors. The ability of the vector system to transfect, induce transgene expression and ultimately induce apoptosis in both RG2 and F98 glioma cell lines was also characterised. In vivo experiments were carried out on cannulated, tumour bearing male Fischer 344 rats. Animals received 6 doses of VLPs delivered intracerebrally every two days with either a low dose (5x107 VLPs per treatment) or high dose (5  108 VLPs per treatment) of IL-12, Empty VLPs or TNE buffer alone. Results: Low dose IL-12 VLP therapy demonstrated a 58% ( 14%) reduction in RG2 tumors compared to controls accompanied by lymphocytic infiltration into the tumor periphery. High dose treatment demonstrated toxicity in both IL-12 (62.5%) and Empty (28.6%) VLP treated animals. Toxicity did not appear to be related to Replication-Proficient Virus (RPV) production or IFN- toxicity. Conclusion: The SFV VLP system has been shown to transfect both glioma cell lines in vitro, mediate transgene expression and induce apoptosis in transfected cells. IL-12 VLP delivery i.c. resulted in a significant reduction in the growth of RG2 tumors while high dose i.c VLP delivery resulted in toxicity. E-mail: [email protected] P 20 Lentiviral gene-silencing vectors and their potential for gene therapy in promyelocytic leukaemia Nicholas Casey ; Greg Woods University of Tasmania, Menzies Research Institute, Hobart, Australia Background: Chromosomal translocations can result in dysregulation of genes involved in cell proliferation and differentiation and are associated with many forms of leukaemia. However these chromosomal breakpoints can result in sequences unique to the cancerous cells, thus presenting targets for the RNA-Interference mechanism, as has been demonstrated previously for a range of target genes. We designed lentiviral-vector delivered shRNA molecules and examined their ability to downregulate the PML-RAR fusion gene in a Promyelocytic Leukaemia (PML) cell line. We also evaluated the vector for potential use in enhancing patient outcomes in the context of autologous transplantation in conjunction with ablative chemotherapy. As the re-

1105 transfusion of non-transduced cancerous or precancerous cells is unacceptable in this context, we also eliminated nontransduced cells, in this case by growth selection. Methods: A series of hairpins designed to target the PMLRAR fusion sequence were tested, with the most promising candidate used for subsequent experiments. A self-inactivating lentiviral vector containing an Internal Ribosomal Entry Site (IRES) was adapted to express both Green Fluorescent Protein (GFP) and Puromycin resistance (PUROr) markers from the same transcript. Target cells were transduced with the anti-PML-RAR hairpin vector, or a nonsense control. After growth selection, a range of assays to determine the effects on the levels of the target gene and protein, as well as assays to determine the effects on cell activity, was performed. Results: Cells treated with the most active anti-PMLRAR hairpin vector exhibited significant knock down of the fusion gene, compared with controls. This correlated with a reduction in cell proliferation. Conclusion: The delivery of active hairpin RNA’s using a lentiviral delivery approach combined with growth selection of transduced cells resulted in the effective control of fusion gene expression and cell proliferation. Planned in vivo studies in immunodeficient mice will further clarify whether this technique is suitable for therapeutic use. E-mail: [email protected] P 21 The histone deacetylase inhibitor FK228 enhances adenoviral transgene expression by a transductionindependent mechanism Angelika Danielsson ; Helena Dzojic ; Wing-Shing Cheng ; Magnus Essand Clinical Immunology, Uppsala University, Uppsala, Sweden The histone deacetylase (HDAC) inhibitor FK228 (also called depsipeptide) is known to induce cell cycle arrest and apoptosis. At lower concentrations, FK228 has also been shown to upregulate CAR expression on certain cell types and this has been claimed to be important for enhanced transgene expression. Here, we observed that FK228 improves adenoviral transgene expression also when administered two hours after adenovirus transduction, indicating a transduction-independent mechanism. We used a vector based on adenovirus serotype 5 (Ad5) and an Ad5 vector with the fiber knob from serotype 35 (Ad5f35) in a variety of human cell lines. Enhanced transgene expression was observed for adenoviral vectors in all cell lines tested. Furthermore, protein expression levels of CAR, CD46 and V5 integrin were not or only marginally upregulated in cells treated with FK228. We also investigated FK228 together with a lentiviral vector. No improved effect was observed on human cell lines but the transgene expression was enhanced in the murine prostate cancer cell line TRAMP-C2 when FK228 was coadministered with the lentiviral vector. It has been shown that valproic acid, another HDAC inhibitor, enhances adenoviral transgene expression but represses replication due to induction of p21. Here, we demonstrate that the replication of a conditionally replicating Ad5 and wild-type Ad5 were not affected by FK228 in the prostate cancer cell line LNCaP. We believe that FK228 can be used in combination with adenovirus therapy to enhance transgene expression and improve oncolytic activity. E-mail: [email protected]

1106

ESGCT 2008 POSTER PRESENTATIONS

P 22

target breast cancer cells via Her2/neu, whose expression is upregulated in a subset of advanced breast cancers. Adenovirus was first chemically conjugated to a polyethylene glycol (PEG) to ablate its promiscuity. Pegylated adenovirus (AdGFP-PEG) showed significantly reduced transgene expression in comparison to the unmodified adenovirus. Next to specifically target Her2/neu expressing breast cancer cells, anti-Her2/neu monoclonal antibody was added to Pegylated adenovirus, generating AdGFP-PEG-HER. As expected, the transduction efficiency of AdGFP-PEG-HER correlated positively with Her2/neu expression. Competition assay with Ad5 fiber protein or Her2/neu antibody confirmed that entry of AdGFP-PEG-HER was mediated via the targeting moiety, Her2/neu antibody. Intravenous administration of Her2/neu-targeted oncolytic adenovirus (YDC002-PEGHER) to MDAMB435 breast tumor-bearing mice elicited greater antitumor effect than either naked Ad or YDC002PEG. The plasma half-life of YDC002-PEG-HER was longer than that of naked Ad, and the liver tropism seemed to be significantly reduced leading to lower toxicity. Moreover, the peak serum IL-6 and TNF- levels was significantly reduced (as much as 70%) in mice treated with YDC002-PEG-HER, revealing that PEGylation can substantially attenuate innate immune responses. In sum, these data demonstrate that active transductional targeting strategies using polymer and antibody can improve the versatility of adenovirus for cancer gene therapy.

Plasmid injection and application of electric pulses alters mRNA and protein expression in B16.F10 mouse melanomas Loree Heller 1; Yolmari Cruz 2; Hong Yang 2; Richard Heller 1 1Old

Dominion University, Center for Bioelectrics, Norfolk, VA, United States; 2University of South Florida, Molecular Medicine, Tampa, FL, United States Purpose: For plasmid gene therapy using electrically mediated delivery, the application of electric pulses to tissues causes cell membrane destabilization, allowing plasmid DNA to enter the cells. Pulse application may result in cell or tissue stress. In order to observe possible regulation of gene expression in response to tumor manipulation, RNA was extracted from mouse B16.F10 melanoma tumors 4 or 24 hours after tumors received plasmid injection alone, two different classes of pulse type, or the combination of plasmid and pulses. Methods: mRNA levels were determined using Stress and Toxicity PCR arrays (SABiosciences, Frederick, MD) for 84 genes in six functional groups. These groups included oxidative and metabolic stress, heat shock, proliferation and carcinogenesis, growth arrest and senescence, inflammation, and necrosis/apoptosis. Individual real-time reverse transcription PCRs were performed to confirm observed changes in mRNA expression. Functional assays, Western blots, or ELISAs were performed to determine resulting changes in protein levels. Results: Using PCR arrays, increases in mRNA levels for several chemokines and cytokines as well as hsp70, iNos, TRAIL and TRADD were observed in response to plasmid injection alone. The increase in inflammatory gene expression in particular confirms previous reports. The application of electric pulses alone also resulted in increased levels of several of these genes as well as glutathione-S-transferase and serpine1. Using individual PCRs for representatives of each mRNA type, increased levels were confirmed for IL1a and TRAIL, while increased levels of protein were confirmed by ELISA for IL1b. Conclusions: Electric pulses may cause short-term increases in some mRNA levels, generally grouped by function into inflammation, heat shock, oxidative and metabolic stress, and necrosis/apoptosis. The levels and kinetics may be related to pulse type, and may also result in augmented protein levels. In particular, an influx of inflammatory cells may account for the increase in some inflammatory gene expression. E-mail: [email protected] P 23 Breast cancer-targeted gene therapy using poly ethylene glycol-modified adenovirus: In vitro and in vivo studies Pyung-Hwan Kim 1; Oh-Joon Kwon 1; Joo-Hyuk Sohn 1; Joo-Hang Kim 1; Seungjoo Haam 2; Chae-Ok Yun 1 1Yonsei University College of Medicine, Institute for Cancer Research, Seoul, Republic of Korea; 2Yonsei University, Chemical Engineering, Seoul, Republic of Korea

Development of novel methodologies to improve specificity and systemic delivery of adenovirus to target cancer tissues continues to be a hot topic of research in enhancing cancer gene therapy. In this study, we aimed to systemically

E-mail: [email protected] P 24 Immunological studies with mouse bcr-abl-transformed cells Vincent Lucansky ; Eva Sobotkova ; Martina Duskova ; Vladimir Vonka UHKT, Department of Experimental Virology, Praha, Czech Republic Chronic myeloid leukaemia (CML) is a malignant disease causally associated with the formation of the Philadelphia chromosome. It develops as a consequence of the reciprocal t(9;22) translocation. The chimeric product of the newly generated bcr-abl fusion gene is known to affect various molecular pathways resulting in malignant transformation. We strongly believe that immunotherapy based on bcr-abl specific immune response could be helpful in the management of this disease. In our experiments we used two mouse (BALB/c) bcr-abl-transformed cell lines, viz B210 and 12B1. In the first series of tests using DNA vaccines based on the 25 amino acid long sequence encompassing the fusion region of the hybrid protein, we failed to elicit sufficient immune respond to protect animals (BALB/c mice) against the challenge with the highly aggressive 12B1 cells. On the other hand, the results obtained after immunisation with a plasmid that carries the whole bcr-abl gene showed that it was possible to induce protection against the challenge with both B210 and 12B1 cells. Subsequent experiments were aimed at producing and testing DNA vaccines based on different fragments of the bcr-abl fusion gene and their combinations. The results indicate that some combinations of these fragments were capable of inducing protection against the 12B1 cells. This work was supported by Grant No. NR9075-3 from the Internal Grant Agency of the Ministry of the Czech Republic. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS

1107

P 25 Gene therapy of human somatotroph and lactotroph pituitary adenomas using an adenoviral virus expressing the somatostatin receptor type 2 Julie Acunzo 1; Catherine Roche 1; Sylvie Thirion 1; Alexandru Saveanu 1; Ginette Gunz 1; Anne Laure Germanetti 2; Bettina Couderc 3; Thierry Brue 4; Alain Enjalbert 1; Anne Barlier 1 1CNRS

UMR 6231 Université de la Méditerranée, Neuroendocrinologie et Neuroimmunologie, Marseille, France; 2Centre Hospitalo-Universitaire Conception, Laboratoire de Biochimie et Biologie moléculaire, Marseille, France; 3Institut Claudius Regaud, Université de Toulouse, Toulouse, France; 4Centre Hospitalo-Universitaire Timone, Département d’Endocrinologie, Marseille, France In human somatotroph adenomas, GH (growth-hormone) hypersecretion can be inhibited by somatostatin agonists such as octreotide. Unfortunately, serum GH levels reach normal values in only 60% of treated patients. The decreased sensitivity to octreotide is strongly related to a lower expression of somatostatin receptor sst2. In this study, the sst2 gene was transferred by an adenoviral vector (Ad-sst2) in human somatotroph and lactotroph adenomas in vitro. Sst2 mRNA levels and sst2 immunostaining dramatically increased after infection. Ten days after infection at 20MOI (multiplicity of infection), sst2 gene transfer decreased cell viability by caspase-dependent apoptosis. At low viral doses (5MOI, a viral dose without effect on cell viability), Ad-sst2 decreased GH or PRL basal secretion and mRNA expression. Somatotroph tumors were classified in 3 groups according to their octreotide sensitivity. After infection by 5MOI Ad-sst2, the maximal GH suppression by octreotide increased in the octreotide partially resistant group and in the resistant ones. In the octreotide-sensitive group, EC50 values significantly decreased without improving maximal GH suppression. Finally lactotroph tumors, non responding to octreotide in basal conditions, became octreotide sensitive. Therefore, sst2 re-expression is able to improve octreotide sensitivity. Because adenoviral vectors can elicit severe inflammatory responses concerns have been raised about the safety of using the first generation of such adenoviral vectors. With a more appropriate vector, our data position sst2 as a new candidate for gene therapy not only for pituitary tumors but also for a wide number of tumors. In fact sst2 gene transfer may open new therapeutic strategies for example in combined treatment with somatostatin analogs or with somatostatin analogs coupled to radioisotopes. E-mail: [email protected] P 26 HDAC inhibitor valproic acid enhances tumour cell kill in adenovirus-HSVtk mediated suicide gene therapy for head and neck squamous cell carcinoma Vishal Kothari 1; Ganesh Joshi 1; Rita Mulherkar

2

1ACTREC,

Tata Memorial Centre, Genetic Engineering, Kharghar, Navi Mumbai, India; 2ACTREC, Tata Memorial Centre, Genetic Engineering, ACTREC, Cancer Research Institute, T.M.C., Khaghar, Navi Mumbai, India Background: Use of adenoviral vectors in various gene therapy trials has been majorly challenged by their low transduction efficiency, an effect attributed to low Coxsackie and

Adenovirus Receptor (CAR) expression on most of the tumor types. Since adenoviral genome has been shown to bind to histone-like proteins, its regulation may be thought to involve activities of histone modifying enzymes such as HDACs. We report that Valproic Acid (VPA), a known Histone Deacetylase Inhibitor, can augment the infectivity and transgene expression from recombinant adenoviruses and helps to achieve better tumor control in a suicide gene therapy approach for Head and Neck Squamous Cell Carcinoma (HNSCC). Methods: NT8e cells were treated with or without 1mM VPA one day prior to AdvHSVtk infection and analyzed on flow cytometer as well as Laser Capture Microscope. RT-PCR analysis of HSVtk, Connexin 43 and 26, Integrin, CAR, and  Actin was carried out in the treated cells. To evaluate prodrug mediated tumor cell kill cytotoxicity was assessed by SRB assay. For in vivo experiments, NT8e HNSCC xenografts established in nude mice were treated with 100l, 10mg/ml VPA one day prior to adenovirus injection, Ganciclovir (2.2mg/ml) was administered for 8 days; and mice were kept under observation for 15 days. Tumor sizes were documented at the end of the treatment. Results: In vitro VPA treatment was found to boost infectivity of AdvHSVtk, expression of CAR, HSVtk, GFP and Connexin 43; as determined by flow cytometry, microscopic and RT-PCR analysis. After infection with suboptimal concentrations of AdvHSVtk followed by GCV treatment, 37.23% cell kill was observed which increased to 75.9% when treated with VPA. In the nude mice xenograft experiments also we observed a dramatic reduction in the tumor volume in vivo. Conclusion: We propose the combined use of VPA, with AdvHSVtk/GCV to achieve better tumor cell kill at reduced virus concentrations. E-mail: [email protected] P 27 Peri-tumoral expression of interleukin-12 is effective against implanted tumors in the liver of syngeneic mice, but achieves only partial protection against distant tumor rechallenge Manuela Gonzalez-Aparicio ; Pilar Alzuguren ; Itsaso Mauleon ; Julien Crettaz ; Gloria Gonzalez-Aseguinolaza ; Jesus Prieto ; Ruben Hernandez-Alcoceba FIMA, Gene Therapy And Hepatology, Pamplona, Spain Background: Interleukin-12 (IL-12) is an immunostimulatory cytokine with a potent antitumor effect demonstrated in different tumor models. Expression of IL-12 needs to be carefully regulated in order to avoid severe adverse effects. We have developed a Mifepristone-inducible system delivered by a High-Capacity adenoviral vector (HC-Ad) in order to obtain regulated production of IL-12 in the liver. Methods: Our tumor model consists on the injection of the murine colon cancer cell line MC38 in the liver of syngeneic C57BL6 mice. The HC-Ad was administered by intra-hepatic injection surrounding the tumors and the inducer Mifepristone was administered by intra-peritoneal injection, following different regimes. Results: We found optimal eradication of hepatic lesions when mice received two cycles of 10 daily inductions with Mifepristone separated 2-3 weeks. Long-term disease-free survival was achieved in most of the cases. In contrast, approximately half of the animals treated with short periods of IL-12 expression died as a consequence of cancer progres-

1108 sion. Mice that were successfully treated by any of the previously described IL-12 regimes were then subjected to a distant (subcutaneous) inoculation of MC38 cells. We observed that tumor progression was reduced in most of the cases compared to naïve controls, but full protection only occurred in approximately 25 % of the animals, irrespective to the initial treatment schedule. Conclusion: Our data suggest that regional expression of IL-12 is effective against hepatic tumors, but establishment of an efficient immunological protection against cancer cells will require further improvements in this immunogene therapy approach. E-mail: [email protected] P 28 Radiosensitization of murine fibrosarcoma tumors by intramuscular mIL-12 gene electrotransfer Simona Kranjc ; Gregor Tevz ; Suzana Mesojednik ; Urska Kamensek ; Maja Cemazar ; Gregor Sersa Institute of Oncology, Department of Experimental Oncology, Ljubljana, Slovenia Background: Nowadays electrogenetransfer (EGT) has become one of promising therapies in treatment of cancer, especially when combined with other treatments. Therefore our study was undertaken to evaluate the antitumor effectiveness of the combination intramuscular murine interleukin-12 (mIL-12) EGT with tumor irradiation (IR) in mouse subcutaneous fibrosarcoma SA-1 and LPB tumors. Methods: Intramuscular mIL-12 EGT was performed by a previously optimized electrotransfection protocol three times, every second day. Subcutaneous tumors (40 mm3) were irradiated locally with the dose of 10 Gy one day after the first application of EGT. Antitumor effectiveness was assessed by tumor growth delay assay and tumors with no tumor growth up to 100 days after the begining of therapy were considered as complete response. Interaction of combined treatment EGT and IR was evaluated by Spector’s formula. Results: The treatment of tumors with intramuscular EGT alone significantly delayed tumor growth compared to the untreated tumors and cured 13% of LPB tumors and 28% of SA-1 tumors. Irradiation alone on SA-1 tumor model only delayed tumor growth, whereas 60% of LPB tumors were in complete response. The combination of EGT and IR of subcutaneous tumors resulted in significant enchancement of tumor complete responses to 44% in SA-1 tumor model and up to 100% in LPB tumor model. Synergistic antitumor effect of the combined treatment was confirmed on both tumor models. Conclusion:The study demonstrates that intramuscular electrogene therapy with IL-12 has good antitumor effect in distantly located subcutaneous sarcoma tumors. Furthermore, gene therapy has synergistic antitumor effect combined with local tumor irradiation, indicating radiosensitization of tumors with IL-12. E-mail: [email protected] P 29 Antitumor therapeutic vaccine employing nonviral genetically modified cells: Role of treg cells Maria Jose Herrero ; Rafael Botella ; Rosa Algas ; Maria Sanchez ; Salvador F. Alino Universidad de Valencia, Farmacologia, Valencia, Spain

ESGCT 2008 POSTER PRESENTATIONS Background: Cancer preventive cellular vaccines have widely demostrated to be succesful even by means of gene therapy nonviral methods, which has been our case. Unfortunately, the translation to therapeutic vaccines has not been able yet to reach the total survival of tumor-bearing treated animals. We present an approach to the understanding of the barriers that block this success. Method: C57Bl/6 mice were challenged with B16 tumor (2  10 4 cells) on “day 0” and 3, 10 and 17 days later were vaccinated with 0,5, 2, Ciclophosphamide 2, or 8 million B16 transfected and irrardiated cells, producing GM-CSF, IL12, B7.2 or combinations of them. Blood samples were taken to study IgG switch and Treg presence. Results. The best tumor growth inhibition was obtained vaccinating with 2 million B16 cells producing GMCSFIL12 and also with GMCSFB7.2. The IgG production, total and subtypes IgG1 and 2a, was always higher than in Control group and reached the maximum values on day 22. Clasical Treg (CD4CD25Foxp3) number was not significantly different in the studied groups. Conclusion: Reaching the success in therapeutic cancer vaccines is getting closer, but more in depth studies of the inhibitory regulatory mechanisms must be performed. At least in our model, classical Treg cells seem not to be involved, on the contrary, another population CD4CD25Foxp3 could be relevant. E-mail: [email protected] P 30 IFN-lambda induces growth suppression of human esophageal carcinoma cells and enhances the sensitivity to chemotherapy Quanhai Li 1; Kiyoko Kawamura 1; Guangyu Ma 1; Nobuo Suzuki 2; Yuki Takei 1; Naoto Yamaguchi 3; Fumi Iwata 4; Muneo Numasaki 4; Hideaki Shimada 5; Masatoshi Tagawa 1 1Chiba

Cancer Center Research Institute, Division of Pathology, Chiba, Japan; 2Graduate School of Medicine, Chiba University, Department of Environmental Biochemistry, Chiba, Japan; 3Graduate Sch of Pharm Sciences, Chiba University, Department of Molecular Cell Biology, Chiba, Japan; 4Faculty of Pharm Science, Josai University, Department of Nutritional Physiology, Sakado, Japan; 5Chiba Cancer Center, Division of Gastroenterological Surgery, Chiba, Japan Novel interferon (IFN)-lambda is consisted of IFNlambda1, -lambda2 and -lambda3, which are also named as interleukin (IL)-29, IL-28A and IL-28B respectively. All three cytokines seem to have similar functions and IFN-lambda is classified as a type III IFN. IFN-lambda could have distinct activities compared with IFN-alpha and IFN-beta although the precise biological properties of IFN-lambda remain uncharacterized. We found that 9 kinds of human esophageal carcinoma cells expressed the receptor complex of IFNlambda consisting of IL-10Rbeta and a novel molecule, IL28R. Recombinant IFN-lambda upregulated the expression level of MHC class I molecules and induced the gene expression of MxA and 2,5-oligoadenylate synthetase, which are involved in anti-viral protection machinery. We also found that IFN-lambda suppressed proliferation of the esophageal carcinoma cells due to multiple mechanisms including cell cycle arrest and apoptosis. The cell cycle arrest was accompanied by the upregulated p21 expression and the increased G0/G1 population. Apoptosis was evidenced by the increased subG1 population and the annexin-V-positive

ESGCT 2008 POSTER PRESENTATIONS cell fraction. Interestingly the inhibited proliferation was not observed in all the carcinoma cells tested although they expressed the IFN-lambda receptor complexes and upregulated MHC class I expression. Adenoviruses bearing the IFNlambda2 gene also induced suppressed growth of the IFN-lambda-sensitive carcinoma cells. We then examined whether IFN-lambda enhanced sensitivity of the carcinoma cells to conventional anti-cancer agents, 5-fluorouracil and cisplatin, and observed additive effects in both agents. In contrast, mesothelioma, pancreatic carcinoma and several normal cells of different origins were negative for the IFNlambda receptors and subsequently were resistant to IFNlambda-mediated growth inhibition. These data suggest tissue specific actions of IFN-lambda and their potentials as an anti-cancer agent in possible combination with chemotherapeutic agents. E-mail: [email protected] P 31 Increased promoter activity is responsible for increased adenovirus transduction efficacy of human laryngeal carcinoma cells to vincristine Dragomira Majhen ; Anamaria Brozovic ; Tvrtko Buger ; Maja Osmak ; Andreja Ambriovic-Ristov Rudjer Boskovic Institute, Division of Molecular Biology, Bijenicka 54, Zagreb, Croatia Adenoviral gene therapy is an approach for treating cancers resistant to currently available therapies such as drug resistant cells. Adenovirus infection occurs via binding of the fiber knob domain to CAR (coxsackie and adenovirus receptor), followed by endocytosis of the virion through interaction of penton base Arg-Gly-Asp (RGD) motif with cellular integrins. On the model of human laryngeal carcinoma (HEp2)-derived vincristine resistant cell line VK2, we observed 3-fold increased wild type adenovirus (Ad5RSVgal) transduction efficacy in comparison with parental HEp2 cells, while for Ad5639RSVgal with short fibers, which infects cells independent of CAR, this difference was 9-fold. We found decreased expression of CAR, increased expression of v3 integrin and equal amounts of v5 integrin in VK2 in comparison to HEp2 cells. In VK2 cells we found slightly smaller amount of internalized virus as well as decreased Ad5RSVgal attachment as compared to HEp2 cells. Finally, Ad5CMVgal, being essentially the same as Ad5RSVgal except for promoter driving expression of transgene, showed equal transduction efficacy in both HEp2 and VK2 cell lines. Therefore we hypothesized that this discrepancy between internalization/attachment and transduction efficacy is likely due to the different activity of RSV and CMV promoters in HEp2 and VK2 cells. We compared the activity of these promoters after transient transfection of plasmids pRSVCAT and pCMVCAT in HEp2 and VK2 cells. In VK2 cells the activity of the RSV promoter was approximately 8-fold higher than in HEp2 cells, while we observed approximately 2-fold increased activity of CMV promoter in VK2 in comparison to HEp2 cells. We conclude that increased promoter activity is responsible for increased adenovirus transduction efficacy of cells resistant to vincristine. Our results indicate that, when evaluating transduction of adenovirus vectors for gene therapy, not only cell surface CAR and/or integrin levels should be measured but also transgene expression driven by different promoters. E-mail: [email protected]

1109 P 32 Construction and development of novel and improved semliki forest virus vectors for RNA-based transient gene therapy Guniz Iskender 1; Sareen E Galbraith 2; Barbara J Kelly 1; Sara J Callagy 1; Brian J Sheahan 3; Gregory J Atkins 1 1Trinity

College Dublin, Microbiology, Dublin, Ireland; University, School of Clinical Sciences, Liverpool, United Kingdom; 3University College Dublin, Veterinary Pathology, Dublin, Ireland 2Liverpool

Background: RNA vectors based on the Semliki Forest virus (SFV) replicon have been developed for gene therapy applications. Replication competent vectors are good candidates for cancer treatments as they would allow longer term therapeutic gene expression and greater penetration of the tumour tissue than suicide virus-like particles (VLPs). However, there is a possibility that unmodified replication competent vectors could enter the central nervous system (CNS) and cause encephalitis. In an attempt to enhance the biosafety of SFV in vivo double deletions were incorporated in nsP3 (TN) and 6k (6k) regions of the genome constructing RSFV TN-6k-26SMCS. To increase the apoptotic activity of SFV, the pro-apoptotic Bax gene was cloned into the replicating vector termed RSFV HA-Bax-26SMCS. Methods: Vectors were studied in vitro and the virulence of the constructs following intramuscular administration was tested in naïve and BALB/c mice immunized with rSFVp62-6k VLPs (recombinant SFV (rSFV) VLPs encoding the p62-6k viral structural proteins). To study the oncolytic effect of the replicating vectors, the murine colon carcinoma cell line, CT-26, was induced subcutaneously in rSFV-p62-6k VLP immunized BALB/c mice. Mice received a total of six direct intratumoural injections of the treatment regimens every two days with a dose of 5  108 p.f.u / 50 l. Results: Vectors displayed a high replication rate in culture and prior immunization with rSFV p62-6k VLPs protected all the mice against the virus produced from the replicating vectors. Treatment of the CT-26 tumours with the vectors resulted in significant inhibition of the tumour growth compared to the negative control. Complete regression of the tumour was observed in 26.6% and 20% of the RSFV TN-6k-26SMCS and RSFV HA-Bax-26SMCS treated mice, respectively. Conclusion: The inherent ability of SFV to induce apoptosis in combination with the enhanced anti-SFV immune response induced by immunization of the mice shows potential for tumour treatment. E-mail: [email protected] P 33 Functional maturation of mouse bone marrow-derived dendritic cells through electroporation of mRNA encoding CD40L, CD70 and caTLR4 Sandra Van Lint 1; Joeri Pen 1; Carlo Heirman 1; Lander Robays 1; Kris Thielemans 2; Aude Bonehill 2 1Vrije

Universiteit Brussel, Department of Physiology and Immunology, Jette, Belgium; 2Vrije Universiteit Brussel, Department of Physiology and Immunology, Laboratory of Molecular and Cellular Therapy, Jette, Belgium Despite the potency of dendritic cells (DC) in priming adaptive immunity, DC-based cancer vaccines have been largely insufficient to effectively reduce tumor burden or

1110 prevent tumor progression in most patients. To enhance DCbased vaccines, we have electroporated DC with mRNA encoding CD40L along with a constitutively active toll like receptor 4 (caTLR4) and CD70, demonstrating the potency of these TriMix DC to generate tumor-specific CTL responses in vitro (Bonehill et al. 2008). As a rigorous preclinical study of this approach, we want to evaluate key parameters of DC activation and function upon TriMix electroporation in vivo. Therefore, we generated C57BL/6 bone marrow-derived DC and matured them with LPS or by TriMix electroporation. Subsequently, the phenotype, cytokine profile and allogeneic T-cell stimulatory capacity of the DC were evaluated in vitro and the DC’s capacity to stimulate SIINFEKL-specific CD8 T-cells was evaluated in vivo. Whereas CD40L alone led to production of IL-12p70 and TLR4 signalling led to production of IL-6/TNF-, their combination resulted in production of all three cytokines. Furthermore, this approach led to expression of DC maturation markers and strong allo-stimulatory capacity of the DC, which was comparable to that of LPS matured DC. Mice, transferred with OT-I cells were immunized with SIINFEKL mRNA electroporated DC, which were either matured with LPS or by TriMix co-electroporation. Seven days after immunisation CD8 T-cells from blood and spleen were analyzed by flow cytometry for their specificity, demonstrating higher numbers of SIINFEKL-specific CD8 T-cells in mice immunized with TriMix DC. These data form the basis for future experiments, where we will evaluate priming of naive epitope-specific CTL and T helper 1 responses, as well as breaking T-cell tolerance against tumor antigens and elimination of pre-established tumors, with a special focus on the contribution of each TriMix component in these processes. E-mail: [email protected]

P 34 The evaluation of angiogenic inhibitory effect of RNA interference in vitro in breast cancer cell lines Ioana Berindan Neagoe 1; Oana Tudoran 2; O Balacescu 2; Loredana Balacescu 1; P Achimas 1; V Cristea 1 1University

of Medicine and Pharmacy, “Iuliu Hatieganu”, Cluj Napoca, Romania; 2Cancer Institute I. Chiricuta, Cluj Napoka, Romania Aim: To examine the effects of several targeted small interfering RNA (siRNA) on proliferation of breast cancer cells in vitro. Methods: Several siRNAs were transfected into human breast cancer cell line HS 578T with Lipofectamine. Cells not transfected with Lipofectamine siRNA served as controls. MTT assay was used to examine the inhibition rate of cell growth. The siRNA transfection was detected by microscopic visualization. The inhibitory effect of siRNA on the expression of VEGF and VEGFR, MMP9 and TNF-alpha mRNAs was detected by RT-PCR. We have evaluated the angiogenic potential of 84 genes using PCR Array technology from Superarray.. Results: siRNA targeting human VEGF, MMP9 and TNFalpha effectively inhibited the proliferation of breast cancer cell line Hs 578T compared with that in control cells. The molecular profile of angiogenesis in breast cancer revealed two groups of genes: overexpressed and inhibited. We have

ESGCT 2008 POSTER PRESENTATIONS obtained designing genes which can be studied to be nominated as angiogenic markers. Conclusion: VEGF, MMP9 and TNF-alpha siRNAs inhibits proliferation of breast cancer cells in vitro. Several genes known as proangiogenic can develop mechanisms for activation of malignant transformation and underline that RNA interference technology is an important tools for molecular inhibition of posttranscriptional gene silencing. E-mail: [email protected]

P 35 Combined autologous bone marrow mononuclear cells and gene therapy in patients with critical limb ischaemia Szyber Przemyslaw 1; Barc Piotr 2; Skora Jan

2

1Medical University of Wroclaw, Vascular Surgery, Wroclaw, Poland; 2Medical University, Vascular Surgery, Wroclaw, Poland

Objective: The search for new options of treatment in critical lower limb ischaemia is of prime and basic importance in vascular surgery. Recent evidence from clinical studies suggests that neovascularization induced by progenitor cells may permit limb salvage. Also gene therapy with the application of plasmid of vascular endothelial growth factor (VEGF) has been conducted in series of successful studies. These two methods may be implemented simultaneously. Our study was designed to investigate and compare the combined bone marrow mononuclear cells and gene therapy to conventional drug therapy in patients with critical leg ischaemia. Materials and Methods: 32 patients with critical leg ischemia persisting for 7 up to 48 months (mean time 27,5) were randomized and divided into 2 groups, each group consisted of 16 patients. In one group administration of stem cells and plasmid was performed while patients out the other groupwere treated pharmacologically with pentoxyphillin. The levels of VEGF in serum and the ankle and brachial indices were measured before, finally 3 months after treatment in both groups. CT-angiography was performed before and after 3 months. Results: Mean (SD) plasma levels of VEGF increased from 238  85 pg/L to the highest vale 387  76 pg/L (p 0.005) 2 weeks after therapy in the experimental therapy group and from 258  55 pg/L to the highest level 376  84pg/L after 28 days of classic therapy. The ankle-brachial index improved from 0.31  0.22 to 0.50  0.23 (p  0.001) in 12 patients (75,0%). In this group CT-angiography showed development of collateral vessels in (68,75%) patients. Ischaemic ulcers healed completely in 11 limbs (68,75%). In the other group the ankle-brachial index did not improve in any patient. Complete healing of skin ulcers was not found in none of the patients. Amputation was performed in 4 (25,0%) patients of the first group and in 6 patients (37,5%) from the second group. Conclusion: These preliminary data after 3 months follow-up indicate that intramuscular injection of mononuclear cells combined with gene therapy in patients with chronic critical leg ischaemia is safe, and more feasible and effective method of treatment than conventional therapy; however, both therapies are limited by a degree of microcirculation. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 36 Hypoxia inducible factor 1 (HIF-1) and HIF-2 additively promote endothelial vasculogenic properties Jeremy Ben-Shoshan 1; Sofia Maysel-Auslender 1; Galia Luboshits 1; Iris Barshack 2; Sylvie Polak-Charcon 2; Eldad Tzahor 3; Gad Keren 1; Jacob George 1 1Sourasky

Tel-Aviv Medical Center, Cardiology, 6 Weizmann St, Tel-Aviv, Israel; 2Sheba Medical Center, Pathology, TelHashomer, Israel; 3Weizmann Institute of Science, Biological Regulation, Rehovot, Israel Background: The transcriptional response of endothelial cells (ECs) to hypoxia is mainly regulated by hypoxia-inducible factor (HIF). HIF-1 and HIF-2 isoforms are structurally similar in their DNA binding and dimerization domains, but differ in their transactivation sites, pointing at differential target genes. We studied the effects of HIF-1 and HIF-2 on the cytokine expression and vasculogenic properties of ECs, as a potential genetic tool to influence angiogenesis. Methods: Two cell lines of ECs (H5V and BAE cells) were transfected to express HIF-1, HIF-2 or both. HIF- constitutive expression was validated at the mRNA and protein levels as well as by assessing the mRNA levels of the HIF direct target genes VEGF and VEGFR2. Dominant negative HIF isoform was used to competitively attenuate HIF activity. The angiogenic activation as well as the paracrine behavior of ECs was investigated by protein arrays. Functional assessment included adhesion to fibronectin and monocytes as well as chemotaxis capacities and tube formation on Matrigel. The paracrine effects of conditioned medium collected from HIF--ECs on the functional properties of wild type ECs was also tested. Finally, vascular formation in vivo was studied by a Matrigel angiogenesis assay in mice. Results: Protein array analysis revealed an altered expression of multiple cytokines in the treated cells, pointing at an additive effect of HIF-1 and HIF-2 on angiogenesis. This trend was also evident on the functional aspect, with regard to the adhesive capacity. HIF-related increased adhesion was found to involve VCAM-1 and ICAM-1 expression in an ERK1/2 dependent manner. Conditioned medium from HIF--ECs paracrinely increased migration and tube formation capacity of wild type ECs, and to a greater extent following HIF-1 and HIF-2 coexpression. Finally, HIF-1 and HIF-2 synergistically promoted vascular formation in vivo. Conclusion: Our results further clarify the separate and mutual roles of HIF-1 and HIF-2 in ECs and suggest potential targets to regulate angiogenic processes. E-mail: [email protected] P 37 Clinical trial with VEGF165 and HGF in patients with severe terminal chronic lower limb ischaemia Andrei Anghel 1; Horatiu Moldovan 2; Alexandru Vasilescu 2; Mihai Ionac 3; Georgel Taranu 3; Edward Seclaman 1; Liviu Tamas 1; Lorand Savu 4 1University

of Medicine and Pharmacy “Victor Babes”, Biochemistry, Timisoara, Romania; 2C.C. Iliescu Institute for Cardiovascular Disease, Cardiovascular Surgery, Bucuresti, Romania; 3Emergency Clinic Hospital Timisoara, Cardiovascular Surgery, Timisoara, Romania; 4GeneticLab SRL, Genetics, Bucuresti, Romania

1111 Background: Nowadays gene therapy has passed the trial phase and is starting to utilize more complex techniques and elements. Transfected angiogenetic factors, especially Vascular Endothelial Growth Factor (VEGF) has proven effectiveness in ischaemic phenomena treatment; modern research is conducted to develop new elements in order to improve the VEGF potential. The present study shows the benefits of Hepatic Growth Factor (HGF) as improving element for VEGF therapy and also application as post-injection anti-inflammatory agent. Methods: We have subclone VEGF165 isoform and HGF into two different pBLAST expression vectors under the control of CMV promoter. Gene transfer was performed in 17 limbs of 15 patients with critical limb ischaemia. A total dose of 1013 copy of both naked plasmids DNA was injected directly into the muscles of the ischemic limb. The clinical evolution has been monitored by ankle-brachial index, walking distance on the rolling carpet, relief of rest pain, Doppler ultrasound and angiography. Results: Intramuscular administration was safe and well tolerated. No tissue edema was noticed. Two months after therapy 12 patients showed partial or complete relief of rest pain, improvement of ischaemic ulcer lesions and increased walking distance on the rolling carpet. The improved perfusion to the distal ischaemic limb was obviated by an increase of more than 0.1 in the ankle-brachial index in 10 limbs. Newly visible collateral vessels are shown by angiography in 8 limbs. Conclusion: Therapeutic angiogenesis with VEGF165 and HGF gene transfer is a safe and non-inflammatory procedure with good effectiveness in the re-vascularisation of the terminal ischaemic lower limb. E-mail: [email protected]

P 38 Heme oxygenase-1 accelerates cutaneous wound healing in mice Anna Grochot-Przeczek 1; Radoslaw Lach 1; Jacek Mis 1; Klaudia Skrzypek 1; Magdalena Kozakowska 1; Patrycja Sroczynska 1; Milena Dubiel 1; Malgorzata Gozdecka 1; Jacek Walczynski 1; Jerzy Kotlinowski 1; Andrzej Mazan 1; Andrzej Rutkowski 1; Krzysztof Kurowski 2; Yann Herault 3; Alicja Jozkowicz 1; Jozef Dulak 1 1Jagiellonian

University, Department of Medical Biotechnology, Krakow, Poland; 2Adamed, Biological Laboratory, Czosnow, Poland; 3CNRS, Centre for Transgenic Animals, Orlenas, France Background: Heme oxygenase-1 is a proangiogenic and cytoprotective enzyme of known anti-inflammatory/immunomodulatory properties. It degrades heme to produce CO, biliverdin and free iron and is induced after blood vessel disruption upon injury. Recently HO-1 has emerged as a potential player in tissue repair. Here we clarify, based on five different murine models, an important role of HO-1 in healing of cutaneous wounds. Methods: The mice were shaved and two full-thickness circular wounds (4 mm in diameter) on each animal were created. The wounds were separated well by 1.5 cm of healthy skin. Each wound was photographed every day. Wound surfaces were measured using ImageJ software and expressed as a percentage of the wound area at day 0. Blood vessels in the wounds were stained for CD31 and analysed under the fluorescent microscope.

1112 Results: First, in wild type mice a competitive inhibition of HO-1 activity with SnPPIX led to impaired skin re-epithelialization and decreased production of many proinflammatory mediators. Second, restoration of tissue integrity was strongly retarded and vascularization was diminished in case of mice lacking one or both HO-1 alleles. Third, mice overexpressing HO-1 in keratinocytes had improved re-epithelialization and vascularization of wounds. Fourth, we observed delayed HO-1 expression and poor vascularization during deregulated wound healing in diabetic (db/db) mice. And finally, we accelerated injury repair and strengthened vascularization of wounds in db/db mice by local overexpression of HO-1 using adenoviral vectors. Conclusion: HO-1 plays an important role in skin wound regeneration. These findings provide new potential approaches in tissue repair. E-mail: [email protected] P 39 Epicardial electroporation as a new method for successful and homogeneous in vivo gene transfer to both atria Olympia Bikou ; Martin Koch ; Ruediger Becker ; Frederik Voss ; Bianca Menrath ; Sina Huntscha ; Hugo A. Katus ; Alexander Bauer University of Heidelberg, Cardiology, Im Neunheimer Feld 410, Heidelberg, Germany Background: Atrial fibrillation is the most common cardiac arrhythmia causing high morbidity and mortality. Currently, pharmacological as well as ablative approaches are the therapy of choice. Both are limited by low efficacy and in some cases severe side effects. Gene therapy might be a viable alternative. However, gene transfer to atrial myocytes is hard to achieve. Simply injecting the recombinant virus into the atrial walls results in low and inhomogeneous infection. In the present study, we investigated a new method to achieve effective and homogeneous infection of atrial myocytes. Methods and Results: In 10 pigs (20  5 kg) two different methods of epicardial injection have been tested. In five pigs adenoviruses encoding for gene fluorescent protein (GFP) were injected into both atrial appendages. The distance between injection sites was 7  2 mm. In another five pigs injection of adenoviruses was followed by electroporation (5 times 20 V/100 ms) applied to both atria. Five days after injection of AdGFP pigs were euthanized and hearts were obtained for further histological examinations. The relation of green fluorescent cells compared to the total number of atrial myocytes was evaluated using fluorescent and phase contrast microscopy (see table below in %, *  p  0.05, RA  right atrium, LA  left atrium, EPO  electroporation).

ESGCT 2008 POSTER PRESENTATIONS result in homogeneous and efficient gene transfer to both atria. Using this technique gene therapy of atrial arrhythmias might be possible. Future studies using specific recombinant adenoviruses affecting atrial electrophysiology are required to evaluate the potentiality of this new therapeutic approach. E-mail: [email protected] P 40 Electrically mediated intradermal delivery of angiogenic growth factors restores perfusion in animal models of peripheral ischemia Bernadette Ferraro 1; Yolmari Cruz 1; Margaret Baldwin 2; Richard Heller 3 1University of South Florida, Molecular Medicine, Tampa, United States; 2University of South Florida, Comparative Medicine, Tampa, United States; 3Old Dominion University, Frank Reidy Research Center for Bioelectrics, Norfolk, United States

Electroporation (EP) is a simple, direct, in vivo method to deliver normally impermeable molecules, such as plasmid DNA, to a variety of target tissues including skin and muscle. In previous work, we evaluated delivery conditions to the skin utilizing a plasmid encoding luciferase. In the current study these conditions were evaluated for delivery of angiogenic growth factors to the skin in areas of ischemia. Therapeutic delivery of angiogenic growth factors is an attractive approach for the treatment of ischemia resulting from a variety of conditions, such as peripheral artery disease and chronic wounds. Other studies have shown that delivery of vascular endothelial growth factor (VEGF) and / or basic fibroblast growth factor (bFGF) successfully increased neovascularization and blood flow but expression levels must be tightly regulated to avoid adverse side effects while still promoting the formation of stable neovasculature. The skin is an attractive target for delivery of plasmid DNA encoding angiogenic growth factors because it allows for enhanced control over expression levels and aids in targeting expression to specific tissue areas. Further, if higher expression levels are needed, the area treated or number of treatments can be increased. To determine the efficacy of this potential non-invasive therapy, two rat models of ischemia were used: a random skin flap model and a hindlimb ischemia model. Specifically, intradermal injection of plasmids encoding human VEGF165 (pVEGF) to the skin flap and bFGF to the ischemic hindlimb followed by EP locally increased expression of both proteins and restored perfusion to the effected area. Further, EP mediated delivery of pVEGF to the random skin flap increased the expression of VEGF regulated angiogenic factors and increased skin flap survival compared to injection of pVEGF alone. E-mail: [email protected] P 41 Effectivity of Adeno-Associated Virus (AAV) mediated gene transfer in isolated porcine and rat cardiomyocytes and in myocardial organotypic culture

Function of both atria was not impaired by electroporation. The amount of inflammation was low. Conclusion: Combining injection of recombinant adenoviruses into the atrial wall with epicardial electroporation

Jan Ksienzyk 1; Matthias Naumer 2; Jürgen A. Kleinschmidt 2; Hugo A Katus 1; Oliver J Müller 1Universitätsklinikum

1

Heidelberg, Internal Medicine III, Heidelberg, Germany; 2German Cancer Research Center, Applied Tumor Virology, Heidelberg, Germany

ESGCT 2008 POSTER PRESENTATIONS

1113

Background: Adeno-Associated virus (AAV) mediated gene transfer into diseased myocardium holds high promises for numerous gene therapy applications. While high transfer efficiencies are achieved in small rodents, gene transfer to the hearts of larger animals is often limited. Aim of our work was development of an ex vivo model to facilitate development of vectors for cardiac gene transfer in large animals and finally patients. Methods: To identify the most suitable ex vivo model, CMV-Luc reporter constructs have been crosspackaged into the capsids of AAV serotype 1,2,6, and 9. These constructs have been tested for their gene transfer efficiency in isolated adult porcine and rat cardiomyocytes and 300m organotypic myocardial slices. Results: Both isolated cardiomyocytes as well as organotypic slices revealed strong transgene expression after transduction with certain serotypes, while other serotypes failed almost completely to mediate transgene expression. Isolated cells and organotypic slices revealed similar results with AAV-6 being most efficient. Comparison with published in vivo data shows similarities for several serotypes, with some differences for others. Comparision between the rat and pig models shows that species differences are also evident ex vivo. Conclusion: The similarity with published in vivo data underlines the feasibility of both models for vector assessment, while remaining differences point to the fact that further refinement should still be worthwhile. The clearly different results between the rat and pig models suggests the employment of human cardiomyocytes and organotypic slices as logical next step.

The fate of Zif-VP64-Ep1 in the cells was studied by Southern Blot and plasmid rescue experiments. eGFP expression was documented by Flow Cytometry and Florescent Microscopy. Real time PCR, Western blotting and Intracellular Flow Cytometry were used to investigate gamma-globin mRNA, gamma-globin protein and HbF protein levels respectively. Binding specificity of the activator was checked by Chromatin Immunoprecipitation (ChIP). Results: Gene transfer was done in K562 and murine betaYAC cells and transfection efficiencies were 65% and 25% respectively. Gamma-globin mRNA levels showed an increase of 250%, gamma-globin protein of 350% and HbF protein of 165%, as compared to the corresponding levels in the untransfected K562 cells, at least 200 generations post-transfection. Zif-VP64-Ep1 was also able to stably transfect murine beta-YAC Bone Marrow cells and, importantly, to activate the expression of the human gamma-globin gene in these cells, as compared to untransfected cells, which do not express gamma globin. Conclusion: Activation of human gamma-globin, by gene transfer of a synthetic activator, is documented. This is the first time that an S/MAR based episomal vector is used for gene transactivation in a cell line and progenitor cells, aiming at specific gene therapy.

E-mail: [email protected]

Aristidis Giannakopoulos 1; Eleana Stavrou 1; Ioannis Zarkadis 1; Adrian J Thrasher 2; Aglaia Athanassiadou

P 42 S/MAR Based episomal vector mediates induction of gamma-globin expression by a specific zinc-finger activator in K562 and murine progenitor cells Eleana Stavrou 1; Eleni Lagadinou 2; Eirini Papapetrou 1; Nicholaos Zoumbos 2; Carlos Barbas 3; Kenneth Peterson 4; Aglaia Athanassiadou 1 1University

of Patras, General Biology, Patras, Greece; of Patras, Hematology, Patras, Greece; 3Skaggs Institute for Chemical Biology, Molecular Biology, La Jolla, California, United States; 4University of Kansas, Biochemistry and Molecular Biology, Kansas City, Kansas, United States 2University

Background: The increase of HbF through the activation of the gamma-globin gene is a valid strategy for the treatment of haemoglobinopathies. The development of a selective, synthetic activator of gamma-globin gene, ZifVP64, based on a zinc-finger DNA binding protein specially designed to bind the gamma-globin promoter 117HPFH area, has been presented (Graslund et al, 2005) showing significant increase of gamma-globin, after viral gene transfer in K562 cells. Additionally, murine cells containing the human beta-globin YAC, expressing beta but not gamma globin can be a good system for screening gamma globin activators (Blau et al 2005). We report the study of an episomal vector of activator Zif-VP64, with the INFB S/MAR supporting retention of episomes in the nucleus of the host cell. Methods: We constructed an episomal vector containing the activator Zif-VP64, the reporter gene CMV-eGFP and the S/MAR element. Gene transfer was done by electroporation.

E-mail: [email protected] P 43 S/MAR’s function in EBV-based episomal vectors for gene therapy is defined by vector backbone sequences 1

1University

of Patras, General Biology, Patras, Greece; Of Child Heath, Molecular Immunology Unit, London, United Kingdom 2Institute

Episomal vectors that replicate extrachromosomally present a safe and attractive alternative to integrating viral vectors for gene therapy applications, as they may be less mutagenic. The Scaffold/ Matrix Attachment Region (S/MAR) of the human beta-interferon gene has been shown to support efficient vector maintenance and transgene expression in mammalian cells, but not in all cases. Identification of factors that predict efficiency of episome retention would therefore be of significant benefit. In cells of haematological origin like Jurkat and K562 cells, DNA plasmid vectors containing EBV-OriP and the EBNA1 gene mediate prolonged gene maintenance, which however, diminishes over time. In order to enhance retention, we combined this system with an S/MAR element. Unexpectedly, this completely eliminated the capacity of episomes to replicate. Calculation of the stress-induced DNA duplex destabilization profile of the vectors suggested that the S/MAR element had created an increase in molecular stability at the OriP site that may have disturbed replicative potential. In contrast, introduction of an alternative Initiation of Replication (IR) region from the beta-globin gene locus instead of EBV-OriP and the EBNA1 expression domain, restored replicative capacity and enhanced episome retention mediated by the S/MAR and these effects were associated with a destabilized region at the IR. These data demonstrate a correlation between S/MAR-mediated vector retention and the presence of an unstable duplex at a replication origin. We consider that the episomal existence of a plasmid requires, apart from defined elements such as the S/MAR, at least one other site of vec-

1114 tor backbone sequence that retains its destabilization potential in the presence of the S/MAR. We therefore suggest that calculations of stress-induced duplex destabilization profiles should be a part of the designing process of S/MAR containing episomal systems E-mail: [email protected] P 44 S/MAR-containing vectors as potential gene therapy episomes Samah Fakhro ; Keith Foster ; Helen Foster ; Capucine Trollet ; George Dickson Royal Holloway University of London, Biomedical Sciences, Egham, United Kingdom The use of naked plasmid DNA as a vector system for gene therapy circumvents many problems associated with the use of viral vectors such as insertional mutagenesis and immune responses. However, the production of a plasmid that replicates as the host cell replicates and remains episomal is still a challenge, and these problems are now being addressed. S/MAR (Scaffold/Matrix Attachment Region) elements are DNA sequences that play fundamental roles in DNA-nuclear matrix attachment, DNA replication, and gene transcription; they could, therefore, have useful applications in gene therapeutic vectors. Several studies have shown that incorporating a S/MAR element in a plasmid allows the vector to persist within replicating cells as an episome for many generations, without integration into the host genome. It also allows continuous transgene expression. The persistence and expression of three plasmids is compared in this study: 1) a conventional plasmid containing a GFP reporter gene 2) a plasmid containing the -IFN S/MAR element (2kb in length) within the open reading frame of the GFP gene and 3) a plasmid containing the Mini-S/MAR (a 0.7kb version of the original S/MAR) also within the GFP open reading frame. Myoblasts and hepatocytes are transfected by lipofection in vitro with the three plasmids described above, followed by selection for 3 weeks, then passaging for a further 10 weeks. Investigations for plasmid maintenance include Plasmid Rescue and PCR; for plasmid integration, Southern Blotting is conducted; and for gene expression, GFP ELISA, Immunofluorescence, and FACS analyses are undertaken. E-mail: [email protected] P 45 Exploiting CD34 myoblast quiescence for the establishment of minicircles and S/MAR-containing episomes in the place of antibiotic selective pressure Samah Fakhro ; Hui Siew Low ; Keith Foster ; Jon Beauchamp ; George Dickson Royal Holloway University of London, Biomedical Sciences, Egham, United Kingdom Minicircles are plasmids with many advantages, including the avoidance of transcriptional silencing due to their lack of bacterial backbone, and their increased safety for use as vectors for gene therapy since they do not contain antibiotic resistance genes.

ESGCT 2008 POSTER PRESENTATIONS S/MARs (Scaffold/Matrix Attachment Region) are elements which, once inserted within a plasmid, allow its persistence as an episome for generations in dividing cells. This ability to be replicated may be due to the element’s high AT base richness that leaves that DNA region in an open conformation, mimicking bacterial Origins of replication. The S/MAR element has also been found to bind to the nuclear matrix, which allows it to remain in the nucleus and segregate as cells divide. If plasmid DNA containing an antibiotic resistance marker is introduced into a cell and remains within it for a certain length of time under selective pressure, cytogenetic changes may occur which lead to the DNA being retained and passed on to daughter cells for generations. This is the basis for the use of selective pressure when a stable cell line is made, which can result in the plasmid integrating into the host genome. However, if DNA is transfected into cells which can then be held in quiescence for 7 days before being allowed to proliferate, the time the DNA remains within the cells’ nuclei may be sufficient for these cytogenetic changes to occur, which eliminates the need for antibiotic resistance markers. Four plasmids are transfected into myoblasts and compared: 1) a conventional plasmid containing a GFP reporter gene 2) a plasmid containing the -IFN S/MAR element (2kb in length) within the open reading frame of the GFP gene, 3) a plasmid containing the Mini-S/MAR (a 0.7kb version of the original S/MAR) also within the GFP open reading frame, and 4) a GFP minicircle construct. The myoblasts are allowed to differentiate, and CD34 myoblasts, akin to satellite cells in vivo, are separated from the myotubes by FACS sorting and held in quiescence for 7 days. They are then allowed to go back into cycle and proliferate. Investigations for plasmid maintenance include Plasmid Rescue and PCR; for plasmid integration, Southern Blotting is conducted;and for gene expression, GFP ELISA, Immunofluorescence, and FACS analyses are undertaken. E-mail: [email protected] P 46 The cellular co-factor LEDGF/p75 determines lentiviral integration sites in the chromosome Zeger Debyser ; Melissa McNeely ; Sofie Vets ; Jan De Rijck ; Koen Bartholomeeusen ; Rik Gijsbers KULeuven, Molecular Medicine, Leuven, Belgium Background: Lentiviruses and the viral vectors derived thereof can insert their viral genome into the chromosome of a non-dividing cell. During this process the viral integrase is assisted by various cellular cofactors. Our group identifies and validates cellular cofactors of nuclear import and integration. LEDGF/p75 was originally identified in our group as a binding partner of HIV-1 integrase and as a chromosomal tether. Data suggest a role for LEDGF/p75 in targeting integration. Methods: LEDGF/p75 was identified to interact with HIV integrase by co-IP. Validation of its role during HIV replication and lentiviral vector transduction was obtained by RNAi knock-down and mutagenesis. Direct interaction between recombinant proteins was demonstrated by pull down and Alphascreen assays. Using eGFP-integrase containing viral particles, viral PICs could be visualized in the nucleus. Results: LEDGF/p75 truncation mutants lacking the chromatin binding domain strongly inhibit HIV replication by competition for interaction with integrase. Stable overexpression of these truncation mutants in cells allowed the selection of HIV strains that overcome the transdominant in-

ESGCT 2008 POSTER PRESENTATIONS hibition. In these resistant strains integrase was mutated at key positions of the LEDGF/p75-integrase interface, providing critical evidence for the importance of LEDGF/p75 in HIV integration. Knock down of LEDGF/p75 severely affected integration sites of lentiviral vectors. The PWWP domain of LEDGF is responsible for the interaction with the chromosome and chimeric LEDGF proteins containing alternative DNA binding domains can substitute for LEDGF. Using Y2H we identified JPO2 and POGz as cellular binding partners of LEDGF/p75. Conclusion: LEDGF/p75 is the cellular tethering factor involved in selection of integration sites. The interaction with integrase is specific for lentivirinae and lentiviral vectors. Our results facilitate the design of safer vectors for gene therapy by fusing the integrase binding domain of LEDGF with sequence specific DNA binding motifs. E-mail: [email protected] P 47 Zinc-finger nuclease induced gene targeting using AAV vectors Katharina Gellhaus ; Christien Bednarski ; Tatjana Cornu ; Regine Heilbronn ; Toni Cathomen Charité Medical School, Institute of Virology (CBF), Berlin, Germany Zinc-finger nucleases (ZFNs) are a promising tool for the precise manipulation of the human genome and therefore of great interest for the treatment of monogenetic disorders. ZFNs comprise an engineered DNA binding domain and the catalytic domain of the FokI endonuclease. Due to their ability to introduce a site-specific DNA double strand break (DSB) within the chosen target locus, ZFNs have been shown to increase the frequency of gene targeting by stimulating homologous recombination (HR)-based DNA repair. Using conventional transfection methods, ZFN-mediated gene targeting can induce gene conversion in up to 20% of cells. Here, we aimed at establishing a ZFN-mediated gene targeting system based on vectors derived from adeno-associated virus (AAV). Due to their ability to transduce multiple tissues, the in vivo or ex vivo delivery of ZFNs via AAV vectors will be a promising alternative. In a proof-of-concept approach, different human cell lines harboring a mutated EGFP-gene were co-transduced with AAV vectors that either served as a donor vector for gene targeting or that contained expression cassettes encoding 2nd generation ZFNs. The frequency of gene targeting and AAV vector integration was assessed by flow cytometry and quantitative PCR on genomic DNA. Titration experiments demonstrated that AAV-based gene targeting is dependent on both the cell line and the vector dose. Using an intermediate AAV vector dose (1–3 x 10e3 gp/cell), EGFP gene conversion was observed in up to 10% of cells. Higher vector doses decreased gene targeting due to the toxicity associated with 2nd generation ZFNs. Assessment of the kinetics of EGFP gene conversion revealed that the plateau was reached 10 days post-transduction, which was significantly slower when compared to DSB-induced gene targeting using an AAV vector expressing the meganuclease I-SceI. Although AAV vector integration was a rare event, PCR-based quantification revealed that the majority of AAV vectors integrated at the nuclease-induced DSB. For (pre)-clinical applications, it will hence be important to further optimize AAV vector design and to reduce the genotoxic side effects by applying 3rd generation ZFNs, which are characterized by optimized DNA binding parameters. E-mail: [email protected]

1115 P 48 Modulation of epigenetic marks controlling the expression of the pancarcinoma antigen epithelial cell adhesion molecule Bernardina van der Gun 1; Sabine Stolzenburg 1; Renata Jurkowska 2; Alice Arendzen 1; Pamela McLaughlin 1; Albert Jeltsch 2; Marianne Rots 2 1University

Medical Center Groningen, Pathology and Medical Biology, Groningen, Netherlands; 2Jacobs University Bremen, School of Engineering and Science, Bremen, Germany

Background: The tumor-associated Epithelial Cell Adhesion Molecule (EpCAM) is a membrane glycoprotein that is over-expressed on most epithelial cancers. High EpCAM expression has been associated with tumorigenic potential, and induced down-regulation of EpCAM decreased cell migration and proliferation. Conventional approaches to downregulate EpCAM, however, have only transient effects. Epigenetic modulation of promoter regions might allow more permanent changes in gene expression levels. Therefore, we set out to identify key epigenetic parameters controlling the EpCAM expression with the goal of rewriting these epigenetic marks to induce a sustained down-regulation of endogenous EpCAM expression. Methods: EpCAM expression was determined by flow cytometry and the DNA methylation status by bisulfite sequencing. The histone code was unraveled by Chromatin Immunoprecipitation Assays (ChIP) performed on EpCAM positive and EpCAM negative human ovarian carcinoma lines. Specific DNA binding molecules were designed by engineering Zinc Finger Proteins (ZFPs) targeting the EpCAM promoter. Inhibition of gene transcription was induced by fusing transient and permanent repression domains to the EpCAM-targeting ZFPs. Results: EpCAM negative cells showed DNA-methylation in the proximal promoter and exon 1, which was absent in EpCAM positive cells. In addition, the histone code of the EpCAM promoter was different in EpCAM positive cells compared to EpCAM negative cells. Induced genome-wide M.SssI-mediated methylation resulted in repression of EpCAM expression, while genome-wide epigenetic drugs like 5-aza-2deoxycytidine upregulated the expression of EpCAM. By targeting the human KRAB or DNA methyltransferase Dnmt3b domains to the EpCAM promoter by ZFPs, over 60% reduction in EpCAM promoter activity was achieved. Conclusion: Epigenetic marks provide insights in the regulation of EpCAM expression. Induced genome-wide reversion of such marks resulted in EpCAM upregulation. The targeted rewriting of epigenetic marks offer new approaches to specifically and permanently interfere with gene expression. E-mail: [email protected] P 49 Targeting of viral vectors in the skin is determined by both extracellular and intracellular cellular factors Haya Falk 1; Nikolai Kunicher 1; Naomi Alvarez-Pereyre 1; Tomer Tzur 2; Amos Panet 1 1Hebrew University-Hadassah Medical School, Virology, Jerusalem, Israel; 2The Hadassah Medical Center, Plastic and Aesthetic Surgery, Jerusalem, Israel

Skin diseases are attractive targets for gene therapy as the target tissue is accessible for the evaluation of efficacy and

1116 toxicity. Understanding viral vector tropism to the different dermal and epidermal human skin cells is a prerequisite for any genetic treatment of skin diseases. Using a novel ex-vivo system of skin organ cultures, that maintain the original organization of the dermis and epidermis layers, we identified by immuno-histochemical techniques the type of cells transduced within the three dimensional organization of the tissue and the factors that determined tropism to the epidermal and dermal cells. We compared in these studies the skin tropism of three different vectors, Adeno-5, Adeno Associated Virus (AAV-2) and Lentivirus-VSV pseudotype, all utilizing ubiquitous receptors. Adeno5 infected mostly early progenitor keratinocyte at the basal layer in between the dermis and the epidermis layers. Limited proteolysis of the skin before infection increased adeno infection at the basal layer as well as at the dermis tissue. AAV2 infected primarily differentiated cells located at the upper layer of the epidermis and lentiviral vectors infected mostly dermal cells. To define restrictions in the epidermal cell layer to lentiviral infection, the mouse epidermal layer was isolated at 48 hrs post infection and virus replication analyzed. Lenti viral reverse transcription was normal in the epidermal cell cytoplasm, as complete linear double strand DNA was detected by PCR analysis. The epidermal restriction to infection appears at migration of the proviral DNA from the cytoplasm as no proviral DNA could be detected in the nuclei, even 48 hrs after infection. Taken together, the results indicated restrictions to viral infection in the intact skin both at the extracellular (Adeno) and intra-cellular (Lentivirus) levels. These restrictions determine viral tropism and would affect any gene targeting of specific skin cells in vivo. E-mail: [email protected] P 50 Identification of a serum factor that can increase adenoviral gene transfer Marko Ahonen 1; Iulia Diaconu 1; Marc Baumann 2; Merja Särkioja 1; Sari Pesonen 1; Akseli Hemminki 1 1University

of Helsinki, CGTG, Molecular Cancer Biology Program &, Transplantation Laboratory, Helsinki, Finland; 2Biomedicum, Protein Chemistry Core Facility, Helsinki, Finland Background: It has been suggested that binding of adenovirus (Ad) to its primary coxsackie-adenovirus receptor (CAR) is an important rate-limiting step for adenovirus delivery. CAR expression in many or most types of advanced solid tumors is quite variable and often low, which may compromise the efficacy of Ad. Also, many normal tissues potentially candidate for Ad gene delivery, such as stem or endothelial cells, are relatively low in CAR. Therefore, it would be advantageous, if Ad gene transfer could be enhanced. In an earlier study assessing the effect of neutralizing antibodies on Ad gene delivery, we found that serum from non-immunized mice, heated to 65°C, increased Ad transgene expression in vitro. The aim of this study is to identify which component in heated serum affects gene transfer. Materials and Methods: Serum was heated to 80°C and cooled prior the infection. Cells were infected with Ad5 which was preincubated in either treated serum or in growth media only. Transgene expression levels were measured by luciferase assay. To evaluate which component in the serum enhanced gene expression, we used Ad5 knob proteins bound to magnetic beads to collect interacting proteins from

ESGCT 2008 POSTER PRESENTATIONS the heated serum. Collected peptides were further analysed by mass-spectrometry. Results: Heated serum significantly increases transgene expression of Ad5 (up to 20-fold) in several cell lines (including A549, UT SCC 29 and M4A4-LM3) compared to unheated serum, serum heated at 55°C or growth media only. With capsid modified viruses, only the CAR binding Ad5 showed the effect, suggesting that the factor interacts with the Ad5 knob specifically. Effect was seen on different mice strains (four out of four) but not with human (three individuals) or bovine serum. Comparison of the proteins collected from heated and unheated serum revealed a particular protein present in the serum only after heating. The protein in question is now being characterized. Conclusion: Factors present in heated mice serum can significantly enhance Ad gene delivery. Isolation and purification of the factor might allow its use as an adjuvant for enhancing Ad gene delivery to low CAR and difficult to infect targets. Also, it might increase our understanding of the interactions of Ad with CAR. E-mail: [email protected] P 51 A lentiviral vector for regulated tat expression activates HIV-1 silent proviruses in latently infected cells David Macías 1; Ricardo Oya 1; Francisco J. SanchezLuque 2; Francisco Martín 2; Francisco Luque 1 1University of Jaen, Molecular studies of human pathologies group, Jaén, Spain; 2Consejo Superior de Investigaciones Científicas, IPBLN, Granada, Spain

Background: Current HAART therapies against HIV-1 infection cannot eradicate the virus because of viral latency of CD4 T-lymphocytes that contain a silent provirus. Latently infected cells represent the most important sanctuary and major molecular obstacle for viral eradication because they can persist for the whole life of the patient. Although, the mechanism that induce the viral latency it is not yet fully understood, there is an absence of Tat protein in cells with latent proviruses. Although some efforts have been made to activate the silent proviruses by the activation of the resting CD4 T cells, the adverse effects and the poor clinical benefit found have shown that this approach is not useful. Method: We have constructed a lentiviral vector that expresses the transactivator tat gene under a promoter controlled exogenously by IPTG to test whether the Tat protein is able to activate silent proviruses. There are potent drugs to block the viral replication in infected patients. However, we have included another element in the vector to increase the safety, the p53 gene is expressed in a Rev-dependant manner, inducing the cell death by apoptosis when the provirus is activated and the blockage of the viral production. Results: The transduction of the CD4 T-lymphocyte cell lines J1.1, ACH-2 and J-Lat GFP clone A72 with this vector shows a viral activation as strong as obtained with TNF or PMA, two described strong inducers of viral activations in these cell lines. Conclusion: These results demonstrate that although the HIV-1 latency is a complex process that involves viral and host factors, including chromatin conformation, miRNAs and other factors, the expression of Tat protein can abolish the latency and activate the provirus. This opens the possibility of targeting this gene to express it in cells harbouring silent proviruses to destroy this reservoir in infected patients.

ESGCT 2008 POSTER PRESENTATIONS E-mail: [email protected] P 52 The generation of animal models to design novel gene correction approaches for Pompe disease Marieke Geel 1; Klari Niezen-Koning 2; Marcel Ruiters 3; Petra Voorn 3; Pamela McLaughlin 1; Alfred Pingout 4; Virginijus Siksnys 5; Marianne Rots 1 1University

Medical Center Groningen, Pathology and Medical Biology, Groningen, Netherlands; 2University Medical Center Groningen, Metabolic Diseases, Groningen, Netherlands; 3Synvolux Therapeutics Inc., Synvolux, Groningen, Netherlands; 4Justus-Liebig-University, Institute of Biochemistry, Giessen, Germany; 5Institute of Biotechnology, Laboratory of Protein-DNA Interactions, Vilnius, Lithuania Background: Pompe disease is a lysosomal storage disease caused by autosomal recessive mutations in the acid-glucosidase (GAA) gene. The GAA deficiency results in glycogen accumulation in the lysosomes, often causing death. The only available therapy is enzyme replacement therapy. Several gene therapeutic approaches can be envisioned for this disorder including gene correction therapy. Unlike Pompe patients, available mouse models show high GAA plasma levels. We developed a mouse model having no/low plasma GAA activities using siRNA. In parallel, DNA binding agents are fused to restriction endonucleases to 1) introduce GAA-mutations resulting in a relevant Pompe animal model and 2) correct the mutated gene in this disease model. Methods: siRNAs against GAA were delivered in fibroblasts using the cationic lipophilic compound SAINT. In BL6 mice, 50g of SAINT  siRNA was injected every day. GAA was determined using 4-MUG assay or mRNA levels. Triplex forming oligonucleotides fused to restriction endonucleases (MunI, scPvuII) and zinc finger nucleases were engineered to target the GAA gene. These so called meganucleases are supposed to introduce double strand breaks into the target gene and thereby increasing the frequency of homologous recombination with an appropriately modified gene fragment supplied together with the meganuclease. Results: Delivery of siRNA decrease GAA activity over 60% in vitro, and with 45% after 14 days in plasma. No reduction of GAA mRNA or protein was found in tissues from siRNASAINT treated mice compared to control mice. Targeted nucleases were constructed and resulted in cleavage of target sequences. Conclusion: Delivery of non-modified siRNA leads to a decrease in plasma GAA activity in vivo and serves as the basis for a clinically relevant Pompe model. This model will then be used to establish the power of engineered gene-specific meganucleases as a new tool in obtaining disease models. E-mail: [email protected] P 53 5 sequence of HMGB2 gene as a tumor specific sequence for transcriptional targeting of glioblastomas Poonam Balani ; Jerome Boulaire ; Ying Zhao ; Jieming Zeng ; Jiakai Lin ; Shu Wang IBN, Gene Delivery, Singapore, Singapore Background: Glioblastoma multiforme (GBM) is a highly invasive brain tumor and one of the most lethal forms of hu-

1117 man cancer (1). Present treatments for GBM, such as surgery, gamma-irradiation, and chemotherapy are ineffective in eradicating the cancer, evidenced by poor prognosis for glioma patients with a mean survival time of less than 1 year after diagnosis. Hence, there is an urgent need to improve the efficacy of therapies for this fatal disease. Method: Identification of the specific sequence by cDNA microarray followed by confirmation using real-time PCR. Cloning of sequence into a baculoviral vector to drive the expression of suicide gene thmidine kinase.Demonstration of in vitro efficacy using cell death assays in presence of ganciclovir and establishment of proof of concept using a mouse xenograft model for gliomas in vivo. Results: HMGB2 was identified as one of the genes having low expression level in normal human astrocytes, but was significantly up-regulated in U251 glioblastoma cells by cDNA microarray analysis. Real-time PCR quantification confirmed 11 to 79 fold increase in HMBG2 expression in glioblastoma tissues vs normal human brain tissue. We cloned a 2kb 5 upstream region of the HMGB2 gene. By progressive truncation of the 2kb fragment, we identified a 500bp fragment displaying very high transcriptional activity in glioblastoma cells, but a low activity in normal brain cells (primary neurons and normal human astrocytes). Baculoviral constructs with 500bp sequence driving the expression of the HSVtk gene were lethal to glioblastoma cells(cell survival 18% after 48Hrs) in the presence of ganciclovir, whereas normal human astrocytes and neurons were not affected. We further confirmed the efficacy of the baculoviral vector in suppressing the growth of human Glioblastoma cells after intra-tumor injection in a mouse xenograft model. Conclusion: We have demonstrated the identification of a novel 5-upstream sequence of the HMGB2 gene which has a potential to be used as an efficient, tumor-selective promoter in targeted glioblastoma gene therapy. E-mail: [email protected] P 54 Adenovirus tropism modification on mesenchymal stem cells infection for in vivo visualization and cancer treatment Carolina Belmar-Lopez 1; Patrick Baril 2; Monica Paya 3; John Overton 4; Rodrigo Dieguez 5; Juan Carlos Ramirez 3; Camino Latorre 1; Georges Vassaux 2; Pilar Martin-Duque 6

1CIBA.

Centro de Investigacion Biomedica de Aragon, Inmunología y Oncología, Zaragoza, Spain; 2INSERM., Biotherapie Hepatique, Nantes, France; 3CNIC. Cento Nacional Investigac. Cardiovasculares, Cardiología Regenerativa, Madrid, Spain; 4Universidad Francisco de Vitoria, Biotechnology, Pozuelo de Alarcón-Madrid, Spain; 5CNIO. Cento Nacional Investigac. Oncológicas, Animales transgénicos, Madrid, Spain; 6Universidad Fco Vitoria/ CIBA. ICS, Biotechnology/ Inmunología y Oncología, Madrid/ Zaragoza, Spain The process of tumour formation is similar to wound healing or tissue regeneration in damaged areas, such that mesenchymal cells proliferate within these sites. On this basis, some groups have suggested that mesenchymal stem cells (MSCs) could serve as vehicles to transport adenoviral vectors specifically to the tumoral area. MSCs have shown to lack the Coxackie Adenovirus Receptor (CAR), which mediates adenoviral infection. However, an alternative mechanism of MSC adenoviral infection was elucidated, whereby viral particles carrying the RGD

1118 peptide on their surface access the cytoplasm via integrinmediated internalisation. A solution to this problem would be to develop vectors expressing surface RGD, but this approach would entail the hugely laborious task of significantly modifying the adenoviral genome. Therefore, we suggest a strategy which overcomes the need for viral manipulation by use of a nanoparticle formulation carrying surface RGD peptide. Our aim was to investigate whether the attachment of the RGD peptide to the adenoviral surface via a nanoparticle formulation (Ad-GFP/RGD-nanoparticle), would enhance viral tropism when compared with a number of alternative strategies. The relative levels of infectivity found when testing the various strategies was measured in terms of the levels of fluorescence detected within the MSCs, after infection with adenovirus encoding green fluorescence protein (Ad-GFP). In vitro, the enhancement of the fluorescence levels of expression from the Ad-GFP/RGD-nanoparticle formulation was noticeable. The mentioned difference was also detected by flow cytometry or by western-blot. Therefore, we conclude that the low levels of adenoviral infection of CAR-negative cell lines such as MSCs, can be overcome by use of the Ad/RGD-nanoparticle system, presumably via exploitation of integrin-mediated internalisation of adenovirus carrying RGD. The Ad/RGD-nanoparticle system offers a promising solution for developing the use of therapeutic adenoviral vectors in cancerous CAR-negative cell lines, without the need for extensive modification of the viral genome. Acknowledgments: Research in authors laboratories were funded by FISs (PI 052626) from the Spanish Ministry of Health, Fundación FIDES. E-mail: [email protected] P 55 Cationic liposome mediated glycotargeting to hepatocytes Moganavelli Singh ; Mario Ariatti University of KwaZulu-Natal, Biochemistry, Durban, South Africa Background: The liver is one of the most important target tissues for gene therapy, playing a central role in metabolism. Gene therapy efficiency will depend largely on the specific targeting ability to liver cells enabling corrective gene transfer. Receptor-mediated gene transfer is a promising approach, provided the relevant receptors are selectively expressed by the hepatocytes. We have used the human hepatoma cell line, HepG2, as a model in vitro gene delivery system. To develop potential gene transfer therapies for liver disorders, HepG2 cells must be successfully transfected. Methods: Four cholesteryl glycosides were prepared for inclusion into a cationic liposome-based system intended for gene targeting to the asialoglycoprotein receptor expressed in HepG2 cells. Cholesteryl--D-galactopyranoside (MSGAL), cholesteryl--D-galactopyranoside (MSGAL), cholesteryl--D-glucopyranoside (MSGLU) and cholesteryl--D-glucopyranoside (MSGLU) were separately formulated with N,N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09), DOPE and pGL3 plasmid DNA into lipoplexes. Liposome:DNA interaction was characterized by band shift, serum nuclease digestion and ethidium displacement assays. Transmission electron microscopy was utilized to determine size and lamellarity of the liposomes and lipoplex size. Growth inhibition and tran-

ESGCT 2008 POSTER PRESENTATIONS sient transfection activities were determined in vitro in the HepG2 cell line. Results: Although all four lipoplexes promoted high levels of luciferase gene expression, the MSGAL system achieved a four fold greater activity than its MSGAL counterpart and an eight fold increase over the MSGLU and MSGLU lipoplexes. The asialoglycoprotein receptor ligand showed a recognition affinity in the order : -galactose galactose -glucose -glucose. An anomeric and an epimeric preference by the asialoglycoprotein receptor for the glycosides is also evident. Conclusion:High targeted gene expression levels coupled to low toxicity in vitro render these novel liposome formulations promising candidates for in vivo studies to be undertaken in the future. E-mail: [email protected] P 56 Optimized vector purification and delivery methods to achieve enhanced liver transduction by AAV vectors Eduard Ayuso 1; Joel Montane 1; Federico Mingozzi 2; Xavier M Anguela 1; Xavier Leon 1; Christopher Mann 1; David Callejas 1; Veronica Jimenez 1; Sabrina Tafuro 1; Anna Andaluz 3; Felix Garcia 3; Shangzhen Zhou 2; Fraiser Wright 2; Katherine A High 2; Fatima Bosch 1 1Universitat Autonoma de Barcelona, Center of Animal Biotecnology and Gene therapy, Bellaterra, Spain; 2Children’s Hospital of Philadelphia, Department of Pediatrics, Philadelphia, United States; 3Universitat Autonoma de Barcelona, Department of Animal Medicine and Surgery, Bellaterra, Spain

Adeno-associated virus (AAV) vectors are promising tools for gene transfer and are able to transduce the liver. However, efficiency of gene transfer to hepatocytes in vivo is generally inadequate due to undesired interaction of vector with extracellular and intracellular compartments. Here we identified different factors that partially contribute to overcome these barriers in vivo. First, we have examined the impact of vector purification on liver transduction. Two AAV1 lots were prepared using either a regular purification protocol or an optimized protocol that allows separating full AAV particles with a high degree of purity. Liver transduction was 10-fold higher in both mice and dogs when using the highly-purified vectors. Similar results were obtained with AAV serotype 8 vectors. Second, we compared standard vs hydrodynamic delivery of AAV vectors by tail vein injection in mouse; a significant increase in transduction was observed after hydrodynamic delivery (up to 30-fold), even in the presence of high-titer anti-AAV neutralizing antibodies. Transduction of AAV may also be affected by the use of genotoxic agents. A dose-response increase in AAV1 transduction of kidney-derived 293 cells and liver-derived HepG2 was observed by adding streptozotocin (STZ) to culture medium. STZ is a potent alkylating agent known to methylate DNA. Surprisingly, we found that IV injection of AAV1 vectors into a STZ-treated dog resulted in transduction of 65% of liver, compared to a 5% observed in non-treated dogs. Streptozotocin is used to induce diabetes in animals and also as a chemiotherapeutic agent in humans. Our results indicate that STZ use should be carefully monitored in the context of AAV-mediated gene transfer. In conclusion, these results may help to decrease vector dose and vector promiscuity, and extend the range of AAV serotypes that can be efficiently used for hepatic-directed gene transfer.

ESGCT 2008 POSTER PRESENTATIONS Supported by SAF2005-01262 and ISCIII (Ciber de Diabetes y Enfermedades Metabólicas Asociadas), Spain and European Community (CLINIGENE, LSHB-CT-2006-018933) E-mail: [email protected] P 57 Widespread correction of murine mucopolysaccharidosis type VII pathology by liver hydrodynamic plasmid delivery Magali Richard ; Audrey Arfi ; Johanne Seguin ; Christelle Gandolphe ; Daniel Scherman INSERM, U640, Chemical and Genetic Pharmacology Laboratory, Paris, France Mucopolysaccharidosis type VII is a lysosomal storage disease caused by deficiency of the acid hydrolase beta-glucuronidase. MPS VII mice develop progressive lysosomal accumulation of glycosaminoglycans within multiple organs, including the brain. Using this animal model, we compared two plasmid gene administration techniques: muscle electroporation and liver-directed transfer using hydrodynamic injection. We have evaluated both the expression kinetics and the biodistribution of beta-glucuronidase activity following gene transfer, as well as the correction of biochemical abnormalities in various organs. This study demonstrates that MPS VII mice treated with a plasmid bearing mouse beta-glucuronidase cDNA, acquires the ability to produce the beta-glucuronidase enzyme for an extended period of time. Liver appeared to be more appropriate than muscle as target organ to enable enzyme secretion into the systemic circulation. A beneficial effect on the MPS VII pathology was also observed, since liver-directed gene transfer led to the correction of secondary enzymatic elevations and to the reduction of glycosaminoglycans storage in peripheric tissues and brain. The present work is one of the first examples showing that non-viral plasmid DNA delivery can lead to both peripheric organs and brain pathology correction in MPS VII mice. It confirms the potential of our systemic gene transfer strategy in neurologic lysosomal disorders. E-mail: [email protected] P 58 Correction of reference memory and synaptic plasticity impairments of pku mice after a single injection of a helper-dependent adenoviral vector expressing pah

1119 zyme, which catalyzes the conversion of phenylalanine to tyrosine. The resultant, untreated hyperphenylalanemia leads to a strong neurologic impairment, whose aetiology has not been fully elucidated but it is probably related to both a reduction of brain amine contents and an attenuation of NMDA receptor function. At present, therapy of PKU relies upon lifelong dietary protein restriction. However, shortcomings in this strategy exist and a more effective cure for PKU is desirable. At this aim, we explored a gene therapy approach focused on the long term correction of the pathologic phenotype of BTBR Pahenu2 mice, the murine model of human PKU. Methods: We produced a helper-dependent adenoviral (HD-Ad) vector expressing the PAH cDNA and administered it to 3-week-old PKU mice, that, before the treatment, showed severe hyperphenylalaninemia. We treated another group of mice with a low phenylalanine (Phe) diet. All mice were subjected to biochemical analyses (Phe determination), behavioural tasks (Morris Water Maze), and synaptic plasticity studies to evaluate the rescue of their pathologic phenotype. Results: We obtained a complete normalization of serum Phe levels in HD-Ad treated mice 2 weeks after the administration of the therapeutic vector and a reversal of PKU-associated coat hypopigmentation. We observed a strong deficit in spatial learning and memory in untreated PKU mice that was completely reversed in HD-Ad treated mice, whereas mice on Phe-restricted diet did not show any amelioration of the phenotype. Studies of synaptic plasticity mirrored this recovery, since we found that long term potentiation was impaired in untreated and diet-treated PKU mice but was restored with our gene therapy approach. Conclusion: In conclusion, our data suggest that gene therapy can be a promising and helpful approach to treat PKU, ameliorating both the biochemical and behavioural pathologic phenotype of the disease. E-mail: [email protected] P 59 Prolonged normoglycemia in STZ-induced diabetic rats transplanted with Adenovirus-mediated human TRAIL gene (Ad5hTRAIL) infected islets Ercument Dirice 1; Ahter Dilsad Sanlioglu 1; Sevim Kahraman 1; Abdulkadir Omer 2; Mustafa Kemal Balci 3; Thomas S. Griffith 4; Salih Sanlioglu 1 1Akdeniz

2Mondino-Tor

University, Faculty of Medicine, Human Gene Therapy Unit, Department of Medical Biology and Genetics, Antalya, Turkey; 2Harvard Medical School, Joslin Diabetes Center, Section on Islet Transplantation and Cell Biology, Boston, United States; 3Akdeniz University, Faculty of Medicine, Division of Endocrinology and Metabolic Diseases, Antalya, Turkey; 4University of Iowa, Gene Therapy Center and Department of Urology, Iowa City, United States

Background: Phenylketonuria (PKU) is one of the most common inborn errors of metabolism; it arises from a deficit in the activity of the phenylalanine hydroxylase (PAH) en-

Background: Type 1 diabetes, characterized by permanent destruction of insulin producing beta cells is lethal unless conventional exogenous-insulin therapy or whole-organ transplantation are employed (Tx). Pancreatic islet Tx is a safer and less invasive method compared to surgery but has been limited by islet graft mal-function and the graft failure. Thus, ex vivo genetically engineered -cells are needed to prolong islet graft survival. TNF-related apoptosis-inducing ligand (TRAIL) plays essential roles during the course of diabetes. Thus, our aim was to develop a novel gene therapy approach by making pancreatic islets over-express TRAIL in

Monica Cerreto 1; Robert Nistico 2; Daniela Ombrone 3; Margherita Ruoppolo 3; Alessandro Usiello 4; Aurora Daniele 5; Lucio Pastore 6; Francesco Salvatore 7 1Ce.In.Ge,

Biotecnologie Avanzate, Gene Therapy, Naples, Italy; Vergata Center, Experimental Neuropharmacology, Rome, Italy; 3Ce.In.Ge Biotecnologie Avanzate, Proteomics and Clinical Proteomics, Naples, Italy; 4Ce.In.Ge, Biotecnologie Avanzate, Neurosciences, Naples, Italy; 5Ce.In.Ge Biotecnologie Avanzate, Molecular Basis of Hereditary Diseases, Naples, Italy; 6Ce.In.Ge Biotecnologie Avanzate, Gene Therapy, Naples, Italy; 7Ce.In.Ge Biotecnologie Avanzate, Biochemistry and Medical Biotechnology, Naples, Italy

1120

ESGCT 2008 POSTER PRESENTATIONS

order to prevent auto-reactive T-cells from attacking the islets. Consequently, we investigated the effect of Ad5hTRAIL-infected islets transplanted into STZ-induced diabetic rats on graft function and survival. Methods: DTZ, PI and FDA staining were employed to determine islet cell purity and cell viability. Transduction efficacy of AdEGFP transduced islets was determined by both fluorometric measurements and fluorescent microscopy. While immunohistochemical methods were performed to show TRAIL expression after Ad5hTRAIL infection, cytotoxic effects of exogenous TRAIL expression were determined with Annexin-V staining. Wistar rats received a single intraperitoneal dose of 30, 40 and 50 mg/kg/BW STZ, and were followed up to 100 days. Ad5hTRAIL and AdLacZtransduced or untransduced rat pancreatic islets were transplanted under the kidney capsule of STZ-induced diabetic rats. The diabetic status after islet TX was followed up for 90 days. Results: AdEGFP infections revealed optimum doses of adenovirus vectors to efficiently transduce pancreatic islets. Ad5hTRAIL infection was not cytotoxic at optimum doses. In terms of blood glucose and survival rates, optimum dose of STZ was 40 mg/kg/BW. The required number of islets transplanted to one recipient was at least 750. While Ad5hTRAIL-infected islets prolonged normoglycemia, AdLacZ-infected pancreatic islets did not manifest such an effect during the 90 day post-Tx period. Conclusion: Notable therapeutic efficiency of Ad5hTRAIL on transplanted pancreatic islets indicates a positive effect of TRAIL over the function and survival of the pancreatic islets.

tive arterioles were also quantified in the skin. Masson’s trichrome staining was performed for the assessment of the collagen organization. Results: Increased total number of capillaries was observed 21 days after AAV-VEGF165 delivery into the wounded area. Moreover, the wound healing rate in animals injected with AAV-VEGF165 was faster than in control mice. Interestingly, the highest rate of re-epithelialization was observed in mice injected with AAV-FGF4-VEGF165 already 13 days after wounding and gene transfer (14,49  3,29% of the initial wound area vs 25,7  2,37% in AAV-LacZ, p  0,05), although there was no angiogenic effect observed in this group at the histological level 21 days after wounding. Moreover, increased collagen deposition was noted after FGF-4, VEGF165 and both genes administration. Conclusion: Simultaneous VEGF and FGF-4 application might represent a new approach to stimulate the reparative processes and improve the elasticity of the wounded skin in diabetes. Supported by grant N302 020 31/1998 and 512/6.PR UE/2008/7 from the Ministry of Science and Higher Education.

E-mail: [email protected]

Stijn Niessen 1; Aiman Mahmoud 2; Ali Aldibbiat 2; Susan Campbell 2; Audrey Brown 2; Chris Huggins 3; Brian Catchpole 4; David Church 1; James Shaw 2

P 60 AAV-mediated VEGF165 and FGF-4 gene transfer for the stimulation of reparative processes in the wounded skin of genetically diabetic mice Agnieszka Jazwa 1; Aleksandra Sierpniowska 1; Magdalena Kozakowska 1; Anna Zagorska 1; Paulina Kucharzewska 1; Justyna Leja 1; Elisa Vahakangas 2; Seppo Yla-Herttuala 2; Alicja Jozkowicz 1; Jozef Dulak 1 1Jagiellonian

University, Department of Medical Biotechnology, Krakow, Poland; 2University of Kuopio, Department of Biotechnology and Molecular Medicine, Kuopio, Finland Background: One consequence of diabetes mellitus is the disruption of the normal process of wound healing, which is related to the decreased production of different growth factors involved in cell proliferation and migration. In this study we assessed the efficacy of the combined gene therapy with two genes, namely human vascular endothelial growth factor-165 (hVEGF165) and fibroblast growth factor4 (hFGF-4) in bicistronic adeno-associated viral vectors (AAV) for the stimulation of reparative processes in the wounded skin of genetically diabetic mice deficient in leptin receptor (Lepr / ). Methods: Two full-thickness longitudinal incisions were made on the dorsum of the mice. Then, 3  1010 of each AAV vector carrying either -galactosidase gene (AAV-LacZ), hVEGF165 (AAV-VEGF165), hFGF-4 (AAV-FGF4) or both VEGF and FGF-4 genes (AAV-FGF4-VEGF165) were injected intradermally into the wound edges of Lepr / mice (n  5/group). The rate of re-epithelialization (wound closure) was measured until all wounds became healed (21 days). Endothelial cells were visualized by binding of Bandeiraea simplicifolia B4 isolectin. -smooth muscle actin(-SMA)-posi-

E-mail: [email protected] P 61 Development of muscle targeted gene therapy with a mutant canine proinsulin plasmid for the treatment of canine diabetes mellitus

1Royal Veterinary College, Veterinary Clinical Sciences, University of London, North Mymms, United Kingdom; 2Newcastle University, Diabetes Research Group, Newcastleupon-Tyne, United Kingdom; 3Newcastle University, Comparative Biology Centre, Newcastle-upon-Tyne, United Kingdom; 4Royal Veterinary College, Pathology and Infectious Diseases, North Mymms, United Kingdom

Background: Development of alternative therapeutic options for canine diabetes mellitus is attractive for various reasons. Firstly, canine diabetes mellitus is common and has been described to be an excellent model of human latent auto-immune diabetes of the adult. Assessment of novel treatment modalities in spontaneously diabetic dogs could therefore provide a useful step towards clinical application in humans. Secondly, a significant number of diabetic pets are euthanased upon diagnosis due to the impact traditional treatment has on pet owner lifestyle, reluctance to perform injections and treatment costs. Muscle-targeted gene therapy (MTGT) employing (pro)insulin constructs provides a potential alternative strategy. This study aimed to develop mutant furin-cleavable canine-specific preproinsulin cDNA and assess its efficacy in vitro and in vivo. Methods: A furin-cleavable canine preproinsulin cDNA (cppI4), designed to yield fully processed mature canine insulin once cleaved by ubiquitous enzyme furin, was de novo synthesised and subcloned into pVR1012null-plasmid. In vitro transfections of murine muscle cells were followed by in vivo transfections. Insulin staining and RT-PCR were used to determine gene expression. Both tibialis anterior muscles of CD1 mice were injected with hyaluronidase and pVR1012-Betagalactosidase or pVR1012-cppI4, followed by electroporation. ELISA-estimation of canine insulin was performed on cellmedium and cell–lysates, serum and muscle homogenates.

ESGCT 2008 POSTER PRESENTATIONS Results: cppI4 functioned adequately in vitro with significant canine insulin biosynthesis and secretion (insulinmedium: 6.72  2.1pmol/l for cppI4; 0.26  0.4pmol/l for Beta-galactosidase, p  0.03 t-test); RT-PCR and staining confirmed insulin expression in vitro. Glucose and body weights were not affected in vivo. Serum and muscle canine insulin was undetectable at 3 and 7 days post-gene-transfer in vivo; Beta-galactosidase-activity assessment proved efficacy of gene-transfer in controls. Conclusion: cppI4 yielded fully processed canine insulin in vitro, but not in vivo. Successful clinical translation requires vector refinement and gene-transfer efficiency enhancement in order to yield significant circulating insulin to be clinically useful. E-mail: [email protected] P 62 Anti-angiogenic and anti-inflammatory gene therapy in diabetic nephropathy Peter Celec 1; Roland Pálffy 2; Michal Behuliak 2; Roman Gardlík 2 1Comenius

University, Department of Molecular Biology, Bratislava, Slovakia; 2Comenius University, Institute of Pathophysiology, Bratislava, Slovakia Background: Angiogenesis and inflammation play an important role in the pathogenesis of diabetic nephropathy. DNA vaccination using human angiomotin (AMOT) effectively inhibited angiogenesis in experimental tumors. Experiments modeling several diseases proved the anti-inflammatory effects of the N-terminal deletion mutant of monocyte chemoattractant protein-1 (7ND). The aim of this study was to analyze the effects of anti-angiogenic and anti-inflammatory gene therapy in a model of diabetic nephropathy. Methods: Male Wistar rats (n  50, age 3 months) were divided into the control group and diabetic group. Diabetes was induced by i.p. injection of streptozotocin. Surviving diabetic rats were divided into DM, 7ND and AMOT groups. After one month 100ug of plasmid DNA (pcDNA3 with either AMOT or 7ND) were injected into hindlimb muscles and electroporated. Renal functions were assessed using metabolic cages and standard biochemistry. After 3 months rats were sacrificed. Markers of oxidative and carbonyl stress were measured in plasma and renal cortex homogenates. Expression of genes related to angiogenesis and inflammation was quantified using real time PCR. Renal histology was analyzed by morphometry of glomeruli. Results: Oxidative and carbonyl stress markers gave inconsistent results. Functional renal parameters were only slightly changed by 4 months of diabetes with glycemia levels over 25 mmo/l. Expression of angiogenic and inflammatory genes, however, was decreased by the respective gene therapy. Glomerular hypertrophy and sclerosis was prevented in both treated groups. Conclusion: Anti-angiogenic and anti-inflammatory gene therapy was effective in preventing diabetic nephropathy. Further studies should focus on potential synergistic effecs. E-mail: [email protected] P 63 Improvement of insulin resistance by adiponectin gene therapy Mei-Hua Nan ; Kyung-Hwan Hwang ; Weiyun Zhang ; Chang-Seon Myung

1121 Chungnam National University, Pharmacology, College of Pharmacy, Daejeon, Republic of Korea Adiponectin (ADN) is an insulin-sensitizing adipokine with low circulating levels in obesity and type 2 diabetes mellitus (DM). It stimulates glucose uptake and fatty acid oxidation and inhibits gluconeogenesis, resulting in an improvement in insulin sensitivity. To examine the ability of ADN to improve insulin resistance, a non-obese type 2 DM mouse model was established by the combined administration of streptozotocin (STZ) and nicotinamide (NA), and plasmid DNA encoding mouse ADN was constructed with cDNA clone of ADN from NIH3T3 cell lines into plasmid vector, pVAX (pVAX/ADN). Subsequently, the pVAX/ADN complexed with Lipofectamine® was transfected into high glucose-induced insulin-resistant cells established in HepG2 cells in vitro and intravenously administered into the type 2 DM mice, and the parameters related with type 2 DM were investigated in vivo. Treatment of 10 nM insulin in insulin-resistant cell line induced the shutdown of insulin signaling measured by the phosphorylation of PKB/Akt which was increased by pVAX/ADN transfection. The administration of pVAX/ADN significantly increased a blood level of ADN and decreased plasma glucose concentration. Moreover, the parameters related with insulin resistance (homeostasis model assessment-insulin resistance, HOMA-IR) and insulin sensitivity (quantitative insulin-sensitivity check index, QUICKI) were significantly improved. Therefore, these results indicated the improvement of insulin resistance by ADN gene therapy, suggesting that this should be a powerful clinical-effective tool for the treatment of metabolic syndrome. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R01-2007-000-20503-0). E-mail: [email protected] P 64 In vivo imaging to detect the duration of adenovirus mediated EGFP transgene expression in allogeneic islet grafts Sevim Kahraman 1; Ercument Dirice 1; Ahter Dilsad Sanlioglu 1; Mustafa Kemal Balci 2; Abdulkadir Omer 3; Thomas Griffith 4; Salih Sanlioglu 1 1Akdeniz

University, Faculty of Medicine, Human Gene Therapy Unit, Department of Medical Biology and Genetics, Antalya, Turkey; 2Akdeniz University, Faculty of Medicine, Division of Endocrinology and Metabolic Diseases, Antalya, Turkey; 3Harvard Medical School, Joslin Diabetes Center, Section on Islet Transplantation and Cell Biology, Boston, Massachusetts, United States; 4University of Iowa, College of Medicine, Center for Gene Therapy, Department of Urology, Iowa City, IA, United States Background: Pancreatic islet transplantation appears to be a promising therapeutic approach for type 1 diabetes. However, inflammation, recurrent autoimmune damage, and islet graft rejection following transplantation are challenging issues to overcome. Therefore, novel gene therapy methods are designed to prolong islet graft survival. For the development and testing of gene therapy approaches determination of transgene synthesis in islet grafts is a crucial step. To determine the duration of adenovirus-mediated reporter gene expression in rat pancreatic islets transplanted into rats CCD (cooled charge-coupled device) camera was used.

1122 Method: Rat pancreatic islets were obtained from Wistar rats and examined for viability and purity and then islets were transduced with adenovirus encoding enhanced green fluorescent protein (AdEGFP) at various multiplicities of infection. Transduction efficiency was determined by fluorescent microscopy and fluorometric measurements. Thirty-six hours after infection, transduced islets were implanted under the left kidney capsule of streptozotocin (STZ)-induced diabetic Wistar recipients. The kidney was exposed through lumber incision post transplantation and the signals from grafts were measured at 1-week intervals using the CCD camera. Results: Our results demonstrated that rat islets can be transduced with AdEGFP at optimum doses without inducing apoptosis and then imaged using CCD camera after implantation into rats . It was observed that in vivo fluorescent signals from grafts could only be detected through direct exposing of kidney to camera because of signal obstruction caused by thick tissue layers. Fluorescence intensities of grafts were gradually reduced with each subsequent week and no signal was detected five weeks after transplantation. Conclusion: Adenovirus provides only a transient gene expression in allogeneic islet transplantation model of type 1 diabetes. In vivo real time fluorescent imaging is a more sensitive and practical method for detecting reporter gene synthesis. This method provides information on the status of islet grafts and allows the monitoring of treatment efficacy in prolonging the graft survival post transplantation. E-mail: [email protected] P 65 Novel furin-cleavable human insulin construct (hppI5) for muscle targeted gene therapy in diabetes mellitus Stijn Niessen 1; Aiman Mahmoud 2; Susan Campbell 2; Audrey Brown 2; Ali Aldibbiat 2; Chris Huggins 3; David Church 1; James Shaw 2 1Royal Veterinary College, Veterinary Clinical Sciences, University of London, North Mymms, United Kingdom; 2Newcastle University, Diabetes Research Group, Newcastleupon-Tyne, United Kingdom; 3Newcastle University, Comparative Biology Centre, Newcastle-upon-Tyne, United Kingdom

Background: Subcutaneous insulin therapy struggles to prevent microvascular complications without hypoglycaemia in diabetes mellitus. Various groups have demonstrated the potential of muscle-targeted gene therapy (MTGT) employing (pro)insulin plasmids yielding constitutive and regulated secretion of (pro)insulin into the systemic circulation without dangerous hypoglycaemia. However, for incompletely understood reasons, furin-cleavage-sites introduction through amino acid substitution, necessary to enable post-translational processing of proinsulin into mature insulin, causes a 10-fold decrease in hormone levels. This study assessed a novel furin-cleavable human preproinsulin cDNA (hppI5) avoiding alterations in amino acids encoding wild type proinsulin/C-peptide to enable production of unmodified mature insulin and C-peptide, as opposed to previously assessed constructs yielding B-10-mutated insulin and truncated C-peptide (hppI4). Methods: hppI5 with furin-cleavage-sites created through addition of amino acids was synthesised and subcloned into pVR1012-null. In vitro transfections of murine muscle cells (C2C12) were followed by in vivo transfections. Insulin staining and RT-PCR were used to determine gene expression. Both tibialis anterior muscles of CD-1 mice were injected

ESGCT 2008 POSTER PRESENTATIONS with hyaluronidase and pVR1012-B-galactosidase, -hppI4 or -hppI5, followed by electroporation. ELISA-estimation of human (pro)insulin and C-peptide was performed on cellmedium, -lysates, serum and muscle. Results: All plasmids functioned adequately in vitro (hppI4: medium-insulin 10.3  1.9pmol/l; hppI5: mediuminsulin: 3.5  1.7pmol/l) with 95% processing into mature insulin; RT-PCR and staining confirmed insulin expression in vitro. Serum and muscle insulin was undetectable at 3 and 7 days post-gene-transfer in vivo. Glucose and body weights did not change. B-galactosidase-activity assessment confirmed efficacy of in vitro/ in vivo gene-transfer in controls. Conclusion: hppI5 yielded fully processed human insulin in vitro. However, it did not outperform hppI4 in vitro or in vivo. The difficulty in attaining bioactive circulating mature insulin through MTGT was re-confirmed. Successful clinical translation requires vector and gene-transfer efficiency enhancement E-mail: [email protected] P 66 Time kinetics of transgene expression in the liver following transduction of neonatal mice with a non-primate lentiviral vector Reba Condiotti 1; Alina Simerzin 1; Michael Themis 2; Simon Waddington 3; Eithan Galun 1 1Hadassah University Hospital, Goldyne Savad Institute of Gene Therapy, Jerusalem, Israel; 2Imperial College London, Gene Therapy Group, London, United Kingdom; 3University College Medical School, Department of Haematology, London, United Kingdom

Background: Previously, we have utilized feline immunodeficiency virus (FIV)-based vectors to efficiently deliver reporter genes to liver cells in adult mice. In a clinical setting, it may be most advantageous to deliver corrective genes at the earliest possible stage in children with metabolic diseases. In order to mimic this situation, neonatal mice were administered FIV-based viral particles and time kinetics of transgene expression in the liver was examined. Methods: One-day-old Balb/c mice were administered via the temporal vein with approximately 3  105 FIV-based viral particles containing the gene encoding luciferase or GFP driven by the CMV or the human alpha1 anti-trypsin (hAAT) liver-specific promoter. At various times post-transduction, transgene expression was examined. Luciferase expression was imaged using a CCCD camera and GFP expression was observed by immunohistochemistry of paraffin-embedded liver tissue sections. Quantitative real-time PCR was used to measure the number of proviral particles contained in genomic DNA extracted from the transduced liver tissue. Results: Luciferase expression driven by the hAAT promoter was stable in 5/6 mice for the duration of the 6-month study whereas the CMV promoter led to weaker expression which decreased with time in 3/5 mice. This phenomenon was most likely due to the known CMV promoter silencing in liver tissue. The percentage of GFP hepatocytes at 2 weeks of age was 3.1  1.3% and increased with time reaching a peak of 17.6  2.3% at 4 months of age. Interestingly, although mice are fully grown at 2 months of age, at which point hepatocytes divide slowly, the number of cells expressing the transgene unexpectedly increased from 8.9  0.7% to 14.4  0.3% between ages 2 and 3 months of age. The pattern of staining revealed clusters of GFP positive cells. Possible explanations of this phenomenon are being investigated.

ESGCT 2008 POSTER PRESENTATIONS Conclusion: Transduction of neonates with FIV-based viral particles leads to efficient transgene expression in liver. E-mail: [email protected] P 67 AAV2-serotype 1, 2, and 8 gene transfer vectors to mediate liver transduction and long-term correction of phenylketonuria following intramuscular injection Alexandre Rebuffat 1; Cary O Harding 2; Zhaobing Ding 1; Beat Thony 1 1University of Zurich, Department of Pediatrics, Steinwiesstrasse 75, Zurich, Switzerland; 2Oregon Health and Science University, Department of Molecular and Medical Genetics, and Department of Pediatrics, Portland, OR, United States

Phenylketonuria (PKU) is caused by hepatic phenylalanine hydroxylase (PAH) deficiency that results in systemic accumulation of phenylalanine (Phe) and its metabolites, and in developmental retardation and intellectual impairment in PKU patients. We have previously demonstrated life-long correction of PKU in the mouse by using a recombinant AAV2 pseudotype-8-mediated transfer of the PAH gene to liver after portal or tail vein injection, and by AAV2 pseudotype-1-mediaed intramuscular administration and expression of PAH plus two essential BH4-cofactor biosynthetic genes (Ding et al. Gene Ther 2006; Ding et al. Mol Ther 2008). Here we report that plasma Phe can successfully be cleared, i.e. from 1.8 mmol/l to below 0.36 mmol/l, also after intramuscular injection of AAV2-PAH vectors pseudotyped with either serotype 8 or 1 with 20-fold or 5-fold lower vector doses than those previously used for tail and portal vein injections, respectively. AAV2-PAH serotype 2 was also investigated but long-term phenotype correction has been observed only for serotypes 1 and 8. Correction was shown in male and female treated mice, albeit more effective in males, as in females, Phe levels began to rise at about 8 months after treatment while in males phenotype correction lasted for the entire period of the experiment ( 1 year). Interestingly, after more than one year all treated females remained only mild hyperphenylalaninemic with Phe blood levels ranging from 0.7-0.9 mmol/l. Supplementation with BH4-cofactor resulted in transient decrease of Phe in all animals that showed a rise in Phe concentration after gene transfer. Alternatively, re-administration of alternate AAV serotypes to avoid immunity against previously administrated viral vectors was also successful for long-term treatment in female PKU mice. Overall, these non-invasive approaches complete our previous study and allow comparing complementary strategies for the development of efficient PKU gene therapy protocols.

1123 Corporation, Genzyme, Framingham, United States; 4University of Oxford, Gene Medicine Group, UK CF Gene Therapy Consortium, Oxford, United Kingdom; 5University of Edinburgh, Medical Genetics Section, UK CF Gene Therapy Consortium, Edinburgh, United Kingdom; 6University of Edinburgh, Medical Genetics Section, UK CF Gene Therapy Consortium, Edinburgh, United Kingdom; 7Imperial College London, Department of Gene Therapy, UK CF Gene Therapy Consortium, London, United Kingdom Firefly luciferase (FLux) is a widely used reporter gene in murine gene transfer studies. In addition to mice, the UK CF Gene Therapy Consortium also assesses gene transfer agents in the sheep lung, because size and cell composition are more similar to the human lung. The cationic lipid GL67, in our hands the most efficient non-viral vector for lung gene transfer, leads to significant levels of FLux expression in the mouse when complexed to plasmids carrying FLux under the control of a CMV promoter (pCIK-FLux). However, we are unable to detect robust levels of FLux in sheep after aerosolisation of GL67A/pCIK-FLux, despite significant expression of vector-specific mRNA (data previously presented). Recently a novel secreted luciferase reporter gene isolated from Gaussia princeps (GLux) has been described (Tannous 2005). We aerosolised sheep with GL67A/pCMVGLux complexes and quantified GLux in bronchoalveolarlavage fluid (BALF), serum, bronchial biopsies and brushings and in lung tissue pieces retrieved at necropsy (n  3 sheep). Compared with controls we detected significant GLux expression in BALF (treated: 6.9  104  5.4  104 RLU/ul, control: 8.1  102  1.4  102 RLU/ul) and lung tissue homogenate (treated: 3.2  104  2.4  104 RLU/ul, control: 3.3  102  1.6  102 RLU/ul). However, GLux expression was undetectable in biopsies and brushings, likely due to the very small amount of epithelial cells in these samples. Expression levels in the sheep lung were not high enough to lead to “spill-over” in serum, which was in contrast to mouse studies where GL67A/pCMV-GLux administration to the lung by nasal sniffing led to significant levels of secreted GLux in serum (treated: 2.9  102  1.5x102 RLU/ul, control: 37.8  14.8 RLU/ul). In conclusion: (1) nonviral gene transfer leads to significant expression of secreted protein expression in BALF, (2) secreted Gaussia luciferase may be a more sensitive reporter gene than firefly luciferase in the sheep lung and allows gene expression to be followed over time by repeatedly sampling BALF fluid. However, it is currently unclear, if GLux is produced by alveolar or airway epithelial cells and sampling techniques have to be refined to address this question. E-mail: [email protected] P 69

E-mail: [email protected] P 68

Optimization of phage C31 integrase system for pulmonary type II cells

Secreted gaussia luciferase is a sensitive reporter of gene transfer in the sheep lung

Manish Aneja ; Rabea Imker ; Michael Kormann ; Senta Uezguen ; Christof Maucksch ; Carsten Rudolph

Uta Griesenbach 1; Catarina C Vicente 1; Ann-Marie Green 2; Seng H Cheng 3; Ian A Pringle 4; Stephen C Hyde 2; Deborah R Gill 2; Gerry McLachlan 5; David Collie 6; Eric WFW Alton 7

Ludwig-Maximilians University, Department of Pediatrics, Division of Molecular Pulmonology, Munich, Germany

1Imperial

College London, Department of Gene Therapy, UK CF Gene Therapy Consortium, London, United Kingdom; 2University of Oxford, Gene Medicine Group, UK CF Gene Therapy Consortium, Oxford, United Kingdom; 3Genzyme

Phage C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. We have successfully reported the activity of this enzyme in murine lungs. However, the final stable long-term expression values observed were presumably too low to correct various inherited lung disorders. The present study aimed

1124 at the optimization of this system for alveolar type II cells. Linear polyethylenimine 22kDa (L-PEI) and Lipofecatime2000 were compared for their transfection efficiency on murine alveolar epithelial cells (MLE12). Though L-PEI and Lipofeactime2000 had similar transfection efficiencies, the resulting long-term expression values obtained with L-PEI were 4.7-fold lower. Release of plasmid DNA from the complexes by adding increasing amounts of heparin sulphate revealed that L-PEI binds to DNA 5-fold more strongly than Lipofectamine2000. Investigations into the effect of promoters, poly A site and a C-terminal NLS revealed no pronounced effects of any of these elements on the integrase activity in type II cells. Different target plasmids were constructed to study their role in achieving stable long-term expression. Long-term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Addition of DNA Methylase and HDAC inhibitors revealed that pEGFPLucattB was silenced post-integration and this phenomenon was presumably responsible for the steep decline in expression from pEGFPLucattB even in the presence of integrase. Improved target plasmids based on previously published pGZCUBI backbone, were constructed with Ubiquitin C and Elongation factor 1 promoters. These improved target plasmids, were immune to postintegrative silencing and with these plasmids, significantly higher stable long-term expression values, compared to those with pEGFPLucattB, could be obtained. The results presented here, in addition to providing evidence for post integrative gene silencing with the ÖC31 integrase system, also demonstrate that efficient expression plasmids when integrated with the ÖC31 integrase, lead to stable long-term gene expression. In this, the integrase presents itself as a promising nonviral gene therapy system for pulmonary disorders. E-mail: [email protected] P 70 Dexamethasone conjugated polyethylenimine as an efficient gene carrier with anti-inflammatory effect for gene therapy of LPS-induced acute lung injury Hyun Ah Kim 1; Sang Hyun Lee 1; Hyunjung Kim 1; Joon Sig Choi 2; Minhyung* Lee 1 1Hanyang

University, Department of Bioengineering, Seoul, Republic of Korea; 2Chungnam National University, Department of Biochemistry, Daejeon, Republic of Korea Acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is caused by disruption of the lung alveolar-capillary membrane barrier. A severe acute inflammatory response in the lung and neutrophilic alveolitis is a hallmark of ARDS/ALI. In this study, dexamethasone conjugated low molecular polyethylenimine (2 kDa, PEI-Dexa) was synthesized as a water-soluble glucocorticoid steroid and a gene carrier for gene therapy of ALI. To confirm the anti-inflammatory effect of PEI-Dexa, PEI-Dexa/p-Luc complexes were transfected into LPS stimulated macrophage cell line (RAW 264.7). After 24hrs, ELISA was performed to measure the levels of TNF-, IL-1, and IL-6. The results showed that PEI-Dexa transfected RAW 264.7 cells secreted less amount of inflammatory cytokines compared to LPS induced control cells. However, PEI 2K/p-Luc and PEI 25K/p-Luc complexes transfected cells did not show this effect. This result indicated that PEI-Dexa had anti-inflammatory effect. Then, PEI-Dexa was characterized as a gene carrier. In vitro transfection and MTT assays showed that PEI-Dexa showed lower

ESGCT 2008 POSTER PRESENTATIONS toxicity to L2 lung epithelial cells than high molecular weight polyethylenimine (PEI 25K). In addition, PEI-Dexa had transfection efficiency comparable to PEI 25K, suggesting that PEI-Dexa is useful for gene delivery to lung epithelial cells. For in vivo study, PEI-Dexa, PEI 2K and PEI 25K/p-Luc complexes were administrated into the mice lungs by intratracheal injection. Saline was used as a control. Gene delivery efficiency of carriers was measured by luciferase assay. The results showed PEI-Dexa had higher delivery efficiency to the mouse lung than PEI 2K or PEI 25K. Finally, in vivo anti-inflammatory effect of PEI-Dexa was evaluated. ALI was induced by intratracheal administration of LPS (0.5mg/kg) in male BALB/c mice. PEI-Dexa/p-Luc complexes were administered to ALI mouse model. After 24hrs, the level of TNF- was determined in BALF and lung tissues. The results indicated that PEI-Dexa reduced the inflammation in the LPS induced ALI. Therefore, PEI-Dexa is a gene carrier with dual-functions of anti-inflammation and gene delivery, and may be useful for gene therapy of acute lung injury E-mail: [email protected] P 71 Comparison of differently pseudotyped lentiviral vectors for gene transfer in the fetal mouse lung Marianne Carlon 1; Jaan Toelen 1; Steffi Mayer 2; Lourenco Sbragia 3; Rik Gijsbers 1; Jan Deprest 2; Zeger Debyser 1 1Catholic

University of Leuven, Laboratory for Molecular Virology and Gene Therapy, Leuven, Belgium; 2University Hospital Gasthuisberg, Department of Gynecology and Obstetrics, Leuven, Belgium; 3University Hospital Gasthuisberg, Department of Pediatrics, Leuven, Belgium Background: For successful in vivo gene transfer of the conducting airways using lentiviral vectors (LV) extra- and intracellular barriers exist. There is a need for an efficient model to screen differently pseudotyped LV to overcome these barriers. Immortalized pulmonary cells (A549) are suboptimal because of lack of apical differentiation. Alternatively, we evaluated a culture model of polarized primary epithelial cells for LV transduction. Since only in vivo testing is conclusive, we validated our findings by fetal intrapulmonary injection of the best performing LV in pregnant mice. Methods: 5 LV were constructed with different envelopes (VSV-G, Ebola, Rabies, Mokola and amphotropic Moloney leukemia virus (Ampho)) encoding both eGFP and firefly luciferase as transgenes. Tracheal cells of adult NMRI mice were isolated and cultured at an air-liquid interface to induce polarization. Subsequently, these cell cultures were transduced after 16d with the above mentioned LV and analysed 72h later by confocal microscopy. In parallel, A549 were transduced with the same LV. Finally, time mated pregnant NMRI mice underwent fetal transthoracal intrapulmonary injection at E17 with the best performing pseudotyped LV or saline. Surviving pups were followed up by bioluminescence imaging (BLI) until 1 postnatal month. Results: In A549, VSV-G and Rabies pseudotyped LV performed best, whereas primary cell cultures were transduced more efficiently by LV with the Mokola, Ebola and Ampho envelope. The latter three were injected transthoracally in the fetal mouse lung. Ebola and Ampho outperformed the Mokola envelope as seen by BLI. Conclusion: Two in vitro models were compared for transduction efficiency with differently pseudotyped LV.

ESGCT 2008 POSTER PRESENTATIONS Since A549 lack apical differentiation, primary polarized cells seem to be a more logic screening tool to test LV. The final aim of comparing different LV pseudotypes, is to find a vector which can apically transduce the conducting airways. The commonly used VSV-G envelope preferentially transduces airway epithelia at the basolateral surface (Wang et al. 1998). In conclusion, Ebola and Ampho pseudotypes emerged as promising in primary cell cultures as well as in vivo and will be further evaluated by fetal intratracheal injection. E-mail: [email protected] P 72 Tight junction openers increase non-viral gene transfer to the airway epithelium in vivo Mia DB Larsen ; Uta Griesenbach ; Eric WFW Alton Imperial College London, Department of Gene Therapy, London, United Kingdom Proof-of-principle for non-viral gene transfer to the lung has been established in several clinical trials. However gene transfer efficiency is low and ways to increase efficiency would be beneficial. Basolateral access of non-viral gene transfer agents to airway epithelial cells may improve cellular uptake and thereby increase gene transfer efficiency. Here, we assessed the effect of the tight junction (TJ) openers EGTA, sodium caprate (C10), sodium laurate (C12) and lysophosphatidylcholine (LPC) on cationic lipid GL67-mediated gene transfer to the airway epithelium in vivo. TJ openers were delivered via “nasal sniffing” to the murine lung and nose, followed by nebulisation of GL67 complexed to a luciferase reporter plasmid (pCIK-Lux). Doses and pretreatment times were optimised for each TJ opener. EGTA did not enhance GL67-mediated gene transfer in the murine airways. However, luciferase expression was significantly (p  0.05) increased in both nasal ( 45 fold) and lung epithelium (3 fold) after administration of C10 (n  810/group). In contrast, C12 and LPC administration increased expression levels significantly (p  0.05) only in the nasal epithelium. The generally more pronounced effect of TJ openers in the murine nose (12-45-fold increase) compared to the lung (1.3-3-fold increase) may in part be related to more effective delivery of the drug to the nose. The nasal epithelium of cystic fibrosis (CF) knockout mice is commonly used to establish proof-of-principle for correction of the CF ion transport defect after gene and small molecule therapy. However, correction with non-viral gene transfer has proven difficult, possible due to low transfection efficiency. Here, we assessed if the 45-fold increase in gene expression after C10-treatment, may allow correction of the ion transport defect. CF-knockout mice were treated with the TJ opener via nasal sniffing followed by perfusion of GL67 complexed to a CFTR plasmid (pCIKCFTR) or a negative control plasmid (n  8-10/group). 48 hr later nasal potential difference measurements were carried out; there was no evidence of altered ion transport. In summary, although C10 pre-treatment significantly increased gene transfer to murine airways, this did not correct the CF defect in knockout mice. E-mail: [email protected] P 73 Aerosol delivery of AAV5-gfp to the rodent lung Ronan Mac Loughlin 1; Brendan Higgins 2; John Laffey 2; Timothy O’Brien 1

1125 1Regenerative

Medicine Institute (REMEDI), National University of Ireland, Galway, Ireland; 2National University of Ireland, Galway, Department of Anaesthesia, Galway, Ireland

Background: rAAV vectors have been shown to be promising vectors in the delivery of therapeutic genes to the lung. Non-invasive aerosol delivery of vector, a key element in therapies for chronic diseases, provides a diffuse, homogeneous distribution pattern with deposition in the smaller airways and alveolar regions. Optimisation of aerosol delivery allows for targeting of vector to areas of the lung but care must be taken in the delivery system design. Factors such as droplet size, effect of aerosol generation and delivery system performance all have an effect on the final therapeutic benefit. Here we describe the characterisation of aerosolised AAV5-gfp delivery to the rodent lung. Methods: Aerosol droplet size and respirable fraction were characterised using laser diffraction. The effect of the aerosolisation on AAV5-gfp was assayed using RT-PCR and flow cytometry. Delivery system performance was determined using spectrophotometric analysis of aerosolised tracer dye extracted from lung. Inflammatory response was characterised using BAL analysis. Results: A droplet size of 3.38  0.02m and respirable fraction (1.5m to 4.5m) of 42.23  0.09% was recorded. A decrease of up to 19% was seen in AAV5-gfp titer, however transduction efficiency on L2 epithelial cells remained unchanged. 29.88  5.73% of original dose of aerosolised Evans Blue was delivered to the lung. BAL cell counts were recorded at approx 29,000 cells/ml and neutrophil differential counts at 5%. Both were within control levels. There was no evidence of inflammation on examination of paraffin embedded lung sections. Conclusion: Generation of an AAV5-gfp aerosol resulted in a respirable fraction that was delivered efficiently to the rodent lung. The effect of aerosolisation was seen to have little effect on the transduction efficiency of the virus and no inflammatory response in the rodent was seen. In conclusion, the work described provides a rationale for aerosol delivery of rAAV to the rodent lung. E-mail: [email protected] P 74 Effects of long-term and global huntingtin silencing Valerie Drouet 1; Valerie Perrin 2; Raymonde Hassig 1; Noelle Dufour 1; Gwennaelle Auregan 1; Sandro Alves 3; Emmanuel Brouillet 1; Ruth Luthi-Carter 2; Philippe Hantraye 1; Nicole Deglon 1 1CEA

- Institute of Biomedical Imaging, Molecular Imaging Research Center, Gif-sur-Yvette, France; 2Ecole Polytechnique Fédérale de Lausanne, SV/BMI/LNGF, Lausanne, Switzerland; 3Faculty of Pharmacy and Center for Neuroscience, Laboratory of Pharmaceutical Technology, Coimbra, Portugal Background: Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (htt) protein. No cure or preventive treatments are available to date to alleviate neurodegeneration. Recent studies have demonstrated that RNAi represents a promising approach for the treatment of autosomal dominant disorders such as HD. But whether an allele-specific silencing of mutant htt or a global silencing could be considered has not previously been addressed and represents a critical issue toward clinical development. Methods: We developed shRNAs targeting mutant and/or wild-type htt transcripts. Lentiviral vectors encod-

1126 ing these shRNAs were injected in the brain of adult rodents and tested for the appearance of a striatal HD pathology. Results: We confirm the therapeutic potential of sihtt administered with lentiviral vectors and showed that initiation of siRNA treatment after the onset of HD symptoms is still efficacious and reduce the HD-like pathology in rodent. We then address the question of the impact of global silencing and demonstrated that silencing of endogenous htt to 2535% in vivo is altering several pathways associated with known htt functions but is not inducing overt toxicity or increasing striatal vulnerability up to 9 months post-treatment. Conclusion: These data indicate that the coincident silencing of the wild-type and mutant htt might be considered as a therapeutic tool for HD. E-mail: [email protected] P 75 The effect of cationic liposome-mediated APOE3 in vivo gene transfer on hippocampal morphology and cognitive status following traumatic brain injury in rats Vadym Biloshytsky 1; Evguen Pedachenko 1; Tatyana Kvitnitskaya-Ryzhova 2; Sergey Mikhalsky 2; Nina Gridina 3; Ludmila Tsyba 4 1Romodanov

Institute of Neurosurgery, Neurotrauma Dept., Kiev, Ukraine; 2Institute of Gerontology, Laboratory of morphology and cytology, Kiev, Ukraine; 3Romodanov Institute of Neurosurgery, Laboratory of experimental neurosurgery, Kiev, Ukraine; 4Institute of Molecular Biology and Genetics, Department of functional genomics, Kiev, Ukraine Background: Apolipoprotein E (APOE, gene; apoE, protein) has been shown to support effective repair and remodeling after neuronal injury, which suggests potential uses of apoE in the treatment of traumatic brain injury (TBI). In this study, we tested the hypothesis that cationic liposome-mediated APOE3 in vivo gene transfer can attenuate morphologic changes and cognitive dysfunction associated with TBI. Methods: Severe diffuse TBI was produced in adult male Wistar rats using the weight drop impact acceleration model (1.5 m/450 g). The mixture of DOTAP liposomes and 25 g of recombinant plasmid pCMV-APOE3 cDNA was infused intraventicularly after TBI using ALZET osmotic pump. Animals (n  20) were randomized into one of four groups: (1) sham-injured and vehicle-treated, (2) brain-injured and vehicle-treated, (3) sham-injured and APOE3-transfected, (4) brain-injured and APOE3-transfected. Results: RT-PCR confirmed the increased expression of APOE3 mRNA in the brain tissue at day 10 after infusion. In 10 days after TBI, CA1 hippocampus exhibited neuronal loss and increase of glial cells number. Neuronal loss was attenuated in APOE3-transfected animals (24% rate of loss as compared to 40% in vehicle-treated rats, p  0.05), gliosis also demonstrated tendency to diminish. Ultrastructurally, gene therapy prevented the evolution of apoptotic-like neurons with chromatin fragmentation, vacuolization of mitochondria in neurons, diffuse axonal damage, destructive changes in myelin fibers, edema of pericapillary astrocytes and microglial activation. Cognitive function was assessed using the Morris Water Maze at day 7-10 after TBI. Brain-injured, vehicle-treated rats demonstrated a profound cognitive deficit (p  0.01) as compared to sham (non-injured) animals. APOE3 gene transfer caused a significant improvement in the learning ability and memory retention, which were different (p  0.05) from injured, non-treated group. Conclusion: Cationic liposome-mediated APOE3 gene

ESGCT 2008 POSTER PRESENTATIONS transfer may have therapeutic potential for the treatment of TBI preventing the evolution of hippocampal neuronal loss and cognitive dysfunction. E-mail: [email protected] P 76 Delivery of heme oxygenase-1 gene using dexamethasone conjugated polyethylenimine for brain protection against ischemic stroke Hyesun Hyun 1; Hyunjung Kim 1; Jaehwan Ryu 1; JaKyeong Lee 2; Minhyung Lee 1 1Hanyang

University, Bioengineering, Seoul, Republic of Korea; University School of Medicine, Anatomy, Inchon, Republic of Korea 2Inha

Dexamethasone is one of anti-inflammatory glucocorticoid steroids. In this research, dexamethasone was conjugated low molecular weight polyethyleneimne (2,000 Da, PEI2k). PEI has been investigated as a gene carrier for gene therapy. Therefore, dexamethasone conjugated PEI (PEIDexa) may have dual functions as an anti-inflammatory reagent and a gene carrier. Heme oxygenase-1 (HO-1) is an anti-apoptotic protein, which can protect cell from apoptosis under hypoxia. In this study, HO-1 gene was delivered to focal ischemic brain of stroke animal model using PEIDexa as a gene carrier. In divided groups of rats (n  5), empty plasmid pNull/PEI-Dexa, pNull/PEI and pSV-HO1/PEI-Dexa complexes were injected directly into the cortex 1 hr prior to middle cerebral artery occlusion (MCAO) except control group. MCAO was performed for 50 min by 40 nylon filament suture into internal carotid artery via the external carotid artery in male Sprague Dawley (SD) rats (280-320g). Also, cortical blood flow was continuously monitored by Laser Doppler Flowmetry. At 24 hrs after MCAO, infarct size was evaluated by 2% 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining. The level of cytokine such as TNF- was measured in the infarcted area. The results showed that injection of pNull/PEI-Dexa reduced infarct size significantly compared with the control and pNull/PEI groups. This suggests that PEI-Dexa protects the brain cells under ischemia-reperfusion condition. The infarct size was further reduced in the group of the pSV-HO-1/PEI-Dexa group, which suggests that HO-1 has synergistic effect with PEI-Dexa in reducing inflammatory response in ischemic brain. Therefore, pSV-HO-1/PEI-Dexa complex is a potential candidate for therapeutic gene delivery to ischemic brain. E-mail: [email protected] P 77 An ultrastructural study of the effect of silencing NG2 inhibitory factor Siobhan McMahon 1; Peter Dockery 1; Eleanor Donnelly 2; John Fraher 3; Padraig Strappe 4; Timothy O’Brien 2 1National University of Ireland Galway, Department of Anatomy, Galway, Ireland; 2National University of Ireland Galway, Regenerative Medicine Institute, Galway, Ireland; 3University College Cork, Department of Anatomy, Cork, Ireland; 4University of Syndey, Center for Brain Research, Sydney, Australia

Lentiviral vectors are an efficient and effective way to deliver therapeutic genes. When spinal cord injury occurs a glial scar is formed resulting in a highly inhibitory environ-

ESGCT 2008 POSTER PRESENTATIONS ment for the growth of axons. This inhibitory environment is mainly due to chondroitin sulphate proteoglycans (CSPG). One of the main inhibitory CSPGs is NG2 and has been shown to be up-regulated in the glial scar following injury. In this study, short hairpin RNAs have been designed to target NG2 and have been cloned into a lentiviral vector. The Neu7 cell line is a rat astrocyte cell line which over expresses NG2, mimicking the inhibitory atmosphere following spinal cord injury. The lentiviral vector containing the shRNA for NG2 was used to transduce Neu7 cells. RNA was extracted from these cells and following reverse transcription PCR 60% knockdown of NG2 was seen in virally-treated Neu7 cells compared to non-treated cells. Many Neu7 cells appeared to stop dividing following lentiviral transduction. In order to identify ultrastructural changes within Neu7 cells following transduction with lentiviral vector expressing shRNA for NG2, Neu7 cells were processed for transmission electron microscope (TEM) analysis. TEM analysis indicated the cells were undergoing ER stress. Stereological analysis was carried out to show ultrastructural changes within the cells. Immunocytochemical staining for markers of ER stress and heat shock proteins appeared to confirm this finding. Changes in cell proliferation following lentiviral vector transduction was found to be related to the unfolding protein response within the cell. E-mail: [email protected] P 78 Gene therapy for peripheral nerve regeneration by Schwann cell-targeting via intrasciatic injection of AAV8 Judit Homs 1; Lorena Ariza 1; Eduard Noguera 1; Gemma Pagès 1; Miguel Chillón 2; Assumpció Bosch 1 1CBATEG,

Biochemistry and Molecular Biology Department, Universitat Autonoma de Barcelona, Bellaterra, Spain; 2ICREA & CBATEG, Biochemistry and Molecular Biology Department, Universitat Autonoma de Barcelona, Bellaterra, Spain Background: Efficient transduction of peripheral nervous system (PNS) is important for the treatment of a wide variety of pathologies including acquired and inherited neuropathies, neuromuscular diseases or pain treatment. Methods: We have characterized the tropism and transduction efficiency of different AAV pseudotypes in primary and established Schwann cell lines as well as in vivo, through sciatic nerve injection in the mouse. Results: Among the AAV pseudotypes tested, AAV2/8 transduced Schwann cells more efficiently, both in vitro and in vivo. On the other hand, AAV2/2 infected preferentially sensory neurons and AAV2/1 transduced both Schwann cells and neurons. Expression of marker genes coded by the different vectors was still present 10 weeks after administration, the overall duration of the experiment. We also analyzed the generation of neutralizing antibodies against AAVs in the infected mice. Antibody titers were higher against AAV1 than against AAV2 or AAV8. Indeed, animals injected with AAV8 showed the lowest titers of neutralizing antibodies against this serotype, correlating with higher expression overtime. To confirm that AAV8 could be a suitable tool to deliver genes into Schwann cells in a mouse model of PNS regeneration, an AAV8 containing cilliary neurotrophic factor (CNTF) was generated. The bioactivity of CNTF was demonstrated in two established Schwann cell lines where its over-expression increased P0 and PMP22 expression, both genes involved in PNS myelin. AAV8CNTF was then administered

1127 into the sciatic nerve of a mouse model of PNS regeneration and we found that, again, P0 and PMP22 myelin proteins were up-regulated 3 weeks after transduction. In contrast to nerves transduced with AAV8GFP, in CNTF transduced mice these results correlated with a significant increase of Gap43 expression in sensory neurons, a marker of growth cone regeneration. Conclusion: These results prove the utility of AAV8 as a gene therapy vector for Schwann cell transduction to treat myelin disorders or to increase regeneration of the PNS. Financed by AFM (AFM2008/13622AE), ISCIII (PI051705) and Generalitat de Catalunya (2006FI00762). E-mail: [email protected] P 79 Tf-lipoplex-mediated c-Jun knockdown results in neuroprotection following excitotoxic injury in the mouse brain Ana Cardoso 1; Pedro Costa 1; Sérgio Simões 2; Luís Almeida 2; Nikolaus Plesnila 3; Carsten Culmsee 4; Ernst Wagner 5; Maria da Conceição Pedroso de Lima 1 1Center

for Neurosciences and Cell Biology, Department of Biochemistry, University of Coimbra, Coimbra, Portugal; 2Center for Neurosciences and Cell Biology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal; 3LudwigMaximilians University, Department of Neurosurgery, Munich, Germany; 4Philipps-University of Marbur, Faculty of Pharmacy, Department of Pharmacology and Toxicology, Marburg, Germany; 5Ludwig-Maximilians University, Department of Pharmacy, Munich, Germany

Background: Non-viral-mediated delivery of siRNAs to the CNS has been restricted by the low levels of tissue distribution and cellular uptake. In this work we report the efficient delivery of siRNA to neurons both in vitro and in vivo via transferrin-associated lipoplexes (Tf-lipoplexes). Methods: DOTAP:Chol cationic liposomes associated with transferrin (Tf) and further complexed with siRNAs against the transcription factor c-Jun were delivered to primary neuronal cultures and to the mouse hippocampus, upon local injection. c-Jun mRNA and protein knockdown mediated by Tflipoplexes were confirmed by QRT-PCR and Western blot, respectively, both in vitro and in vivo. Astrocyte proliferation and microglia activation were evaluated by immunohistochemistry using the GFAP and CD11b cell markers. Results: Approximately 70% reduction of c-Jun mRNA levels and 50% reduction of c-Jun protein levels, were achieved 48 h after transfection with Tf-lipoplexes, without significant cytotoxicity. c-Jun knockdown resulted in significant protection from glutamate damage or oxygen-glucose deprivation, in primary cultures from cortical neurons, and in an impressive neuroprotective effect in an in vivo model of excitotoxic injury. Cresyl violet staining indicated that Tflipoplex-mediated c-Jun knockdown reduced neuronal death in the CA3 region of the mouse hippocampus when c-Jun siRNA delivery was performed immediately after or 3 days before kainic acid injection. Moreover, a significant decrease in the number of astrocytes and microglia activation was observed upon c-Jun silencing. The anti-inflammatory effect of this strategy was further demonstrated by the decrease of pro-inflammatory cytokine levels following Tflipoplex injection. Conclusion: Overall, our results demonstrate that Tflipoplexes mediate efficient gene silencing in neuronal cells both in vitro and in vivo, with minimum toxicity, which may

1128 prove useful for both target validation and possible new therapeutic approaches to neuronal repair. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 81 AAV-mediated PEDF gene transfer to a transgenic mouse model of diabetic retinopathy Virginia Haurigot 1; Pilar Villacampa 1; Albert Ribera 1; David Ramos 2; Bosch Assumpció 2; Jesús Ruberte 1; Fàtima Bosch 1

P 80 Development and validation of quality control release tests for 3rd generation lentiviral vectors for clinical applications Giuliana Vallanti 1; Francesca Rossetti 1; Laura Sonzogni 1; Francesca Bellintani 1; Giuseppina Marano 1; Chiara Pozza 1; Francesca Salvatori 1; Claudio Bordignon 2; Luigi Naldini 3; Marina Radrizzani 1 1MolMed SpA, Development, Milan, Italy; 2MolMed SpA, President & CEO, Milan, Italy; 3San Raffaele Scientific Institute, Telethon Institute for Gene Therapy, Milan, Italy

MolMed S.p.A. has developed a large scale production and purification process for 3rd generation lentiviral vectors to be used in phase I/II clinical trials. Innovative quality control tests have been developed for vector characterization. A full QC plan has been designed for the release of clinical lots, in accordance with CFR21 GMPs, CBER Points to Considerer and ICH. The tests selected for assessing identity/potency, safety and purity are summarized in the table below, along with the corresponding specifications. All safety tests, ELISA to detect Large T Antigen (LTA) and Real Time PCR to detect LTA and E1A genes have been fully validated.

1Universitat

Autònoma de Barcelona, Center for Animal Biotechnology and Gene Therapy, CIBER Diabetes y Enfermedades Metabólicas Asociada, Bellaterra (Barcelona), Spain; 2Universitat Autònoma de Barcelona, Center for Animal Biotechnology and Gene Therapy, Bellaterra (Barcelona), Spain Diabetic retinopathy is the leading cause of loss of visual acuity and blindness in adulthood. Transgenic mice overexpressing IGF-I in the retina constitute an excellent animal model of human diabetic retinopathy. In these mice, retinopathy progresses from a non-proliferative phase characterized by breakdown of the blood-retinal-barrier, thickening of the basement membrane and loss of pericytes in vessels of young animals to an openly proliferative retinopathy, with neovascularisation and eventual retinal detachment, which makes these mice very suitable for assaying new therapeutic approaches. Current treatments for diabetic retinopathy, such as laser photocoagulation and vitrectomy, are invasive and merely palliative and repetitive local delivery of drugs is not desirable in a chronic disease. Viral-vector-mediated gene transfer allows continuous local production of therapeutic proteins. Our studies after intravitreous delivery of adeno-associated type 2 viruses coding for Green Fluorescent Protein (GFP) have shown that, in our animal model of retinopathy, AAV2 has a high efficiency in the transduction of cells of the ganglionar layer and also cells in the inner nuclear layer. With the aim of overcoming the angiogenic process present in these mice, we designed a strategy for the continuous production of Pigmented Epithelium Derived Factor (PEDF) using an AAV2 viral vector. PEDF is normally secreted by retinal pigmented epithelium cells and its levels are decreased in the vitreous humour of diabetic patients. PEDF is a potent antiangiogenic factor and also has neuroprotective properties, making it an excellent candidate for gene transfer in diabetic retinopathy. PEDF was detected in retina and aqueous humour samples from IGF-I mice six months after intravitreal injection of AAV2PEDF. PEDF-treated IGF-I mice had a significant reduction in the neovascularisation of the retina, as measured by fluorescein angiography morphometry, and also showed a decrease in the expression of the retinal inflammatory marker Intercellular Adhesion Molecule 1 (ICAM-1). These results indicate that AVV-mediated PEDF gene transfer to the eye may constitute a feasible approach for the treatment of neovascularisation in diabetic retinopathy. E-mail: [email protected] P 82 Functional rescue in a mouse model for autosomal dominant Retinitis Pigmentosa following mutationindependent gene therapy

Sophia Millington-Ward ; Naomi Chadderton ; Arpad Palfi ; Mary O’Reilly ; Peter Humphries ; Paul F. Kenna ; G. Jane Farrar Trinity College Dublin, Genetics, Dublin, Ireland E-mail: [email protected]

Whereas gene therapies for recessive retinal disorders have made great progress and reached phase I clinical trial,

ESGCT 2008 POSTER PRESENTATIONS therapies for autosomal dominant disease have lagged behind, frequently due to their inherent complexity. Therapies for many dominant disorders may require suppression of mutant alleles. However, these disorders frequently show immense mutational heterogeneity. Hence, for such disorders a mutation-independent suppression and replacement gene therapy has been proposed based on the redundancy of the genetic code. Rhodopsin-linked Retinitis Pigmentosa (RP) is a genetic disease that leads to photoreceptor cell death and thereby loss of vision. Utilising AAV-mediated mutation-independent gene therapy, we demonstrate functional rescue in a mouse model for autosomal dominant RP (adRP), the Pro347Ser mouse carrying a human mutant rhodopsin gene. Resulting histological and electrophysiological (ERG) data are presented. Notably, these represent the first data showing functional improvement following gene therapy in an animal model for adRP. E-mail: [email protected]

P 83 Gene therapy of experimental autoimmune encephalomyelitis using lentiviral vector expressing vasoactive intestinal peptide (VIP) Marien Cobo 1; Per Anderson 1; Pilar Muñoz 1; Miguel Toscano 2; Nieves Varela 1; Mario Delgado 1; Francisco Martin 1 1IPBLN,

CSIC, Immunology and Cell Biology Department, Armilla , Granada, Spain; 2Temple University School of Medicine, Department of Microbiology and Immunology, Philadelphia, United States Background: Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), an autoimmune demyelinating process of the CNS. The vasoactive intestinal peptide (VIP) is a very potent therapeutic agent for several autoimmune disorders in animal models. However, VIP is very unstable and daily injections are needed to see an effect. In this work we use Lentiviral vectors expressing VIP as an alternative to VIP peptide to treat EAE. Methods: We use a chronic model of EAE using MOG3555 in C57/BL6. Treatment consisted in a unique intra-peritoneal injection of lentiVIP (a SIN lentiviral vector expressing VIP cDNA through the CMV promoter) at different points of disease progression. Intracellular cytokine staining, proliferation and cytokine secretion upon MOG stimulation of DLN were use to measure immune modulation. Results: LentiVIP injection not only prevents disease progression but also reduce EAE score when injected at peak of disease. MOG-Proliferative response of DLN cells of LentiVIP-treated mice was diminished compared to EAE control mice. We also show a slight increase of regulatory T cells population and higher levels of IL17. Conclusions: In vivo administration of lentiVIP results in a more prominent disease reduction than VIP peptide daily injections. E-mail: [email protected]

1129 P 84 Delayed GDNF gene delivery in the striatum of 6OHDA-treated rats increases tyrosine hydroxylase without correlation with dopamine levels Xin Yang 1; Birgit Mertens 2; Enni Lehtonen 1; Linda Vercammen 3; Olivier Bockstael 1; Abdelwahed Chtarto 1; Marc Levivier 1; Jacques Brotchi 1; Yvette Michotte 2; Sophie Sarre 2; Liliane Tenenbaum 1 1Université

Libre de Bruxelles, Lab. Exp. Neurosurgery, Brussels, Belgium; 2Vrije Universiteit Brussel, Department of Pharmaceutical Chemistry, Drug Analy, Brussels, Belgium; 3Katholieke Universiteit Leuven, Laboratory for Neurobiology and Gene Therapy, Leuven, Belgium A tetracycline (tet)-inducible AAV serotype 1 vector expressing human GDNF cDNA (AAV1-tetON-GDNF) was administered in the striatum of rats 5 weeks after lesioning by intrastriatal 6-hydroxydopamine injection. A significant tet-dependent improvement of motor symptoms was evidenced 4 weeks post-vector injection. At later time points, a partial behavioural recovery was observed in all groups but no further improvement could be evidenced in GDNF-treated animals. Fourteen weeks post-vector injection, tyrosine hydroxylase (TH) levels were significantly higher and striatal THpositive reinnervation significantly increased in GDNF-treated rats but the striatal dopamine content remained unchanged. We show that the lack of functionality of the additional THpositive fibers in GDNF-treated animals could be due to a defect in TH phosphorylation. In addition, the TH increase required long-term GDNF overexpression since withdrawal of tet after 4 weeks, resulted in TH levels comparable to the control groups. These data suggest that delayed GDNF gene delivery stimulates some components of dopamine metabolism but does not rescue dopaminergic neurons. E-mail: [email protected] P 85 Evaluation of small epitope tags for immunohistochemical detection of transgene expression in vivo Evy Lobbestael ; Kirsten Paesen ; Chris Van den Haute ; Rik Gijsbers ; Zeger Debyser ; Veerle Baekelandt ; JeanMarc Taymans Catholic University Leuven, Molecular medicine, Leuven, Belgium Background: The transgenic overexpression of proteins in vivo is a powerful approach to study the basic biology of proteins, to generate disease models or to evaluate gene therapy approaches. When investigating a transgenic protein, a specific and sensitive detection is indispensable. Unfortunately, antibodies raised against the protein of interest are not always available. It is also necessary to discriminate endogenous from overexpressed proteins. The use of an epitope tag fused to the protein may circumvent these difficulties. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags. In the present study, we evaluated several small epitope tag/antibody combinations for the detection of overepxressed proteins in cell extracts as well as in intact rodent brain, using eGFP as a reference. Methods: We fused different epitope tags (AU1, flag, 3flag, HA, myc and V5) N-terminally to eGFP and made lentiviral vectors with these constructs. After transduction of

1130 HEK293T cells, we analysed the different constructs via SDSPAGE. Using the same lentivectors, stereotaxic injections were performed in the striatum of Wistar rats. After staining the vibratome sections with the appropriate tag antibodies, we examined the size of the transduced area and the number of transduced cells recognised by the tag antibody compared to eGFP detection. Results: We found that all the examined antibodies can detect the tag-eGFP fusion protein in cell extracts as well as in vivo, in varying degrees. The anti-V5 antibody seems to detect the V5-eGFP in a very sensitive manner, both on western blotting and in vivo. Currently, we are further investigating the sensitivity and specificity of these epitope tag/antibody combinations in vivo. Conclusion: The selection of an appropriate epitope tag provides the possibility to have an unambiguous detection of any overexpressed gene of interest. E-mail: [email protected] P 86 Comparative study of different recombinant viral vectors for transduction of dopaminergic neurons in rodent brain Anke Van der Perren ; Jaan Toelen ; Chris Van den Haute ; Zeger Debyser ; Veerle Baekelandt Catholic University Leuven, Molecular Medicine, Leuven, Belgium Background: Both lentiviral vectors (LV) and recombinant adeno-associated viral (rAAV) vectors are frequently used for gene transfer into the central nervous system. For rAAV, several new serotypes have recently been described with varying tropism for different brain regions. Earlier studies described neuronal expression of several rAAV serotypes, but a comparative study on the transduction efficiency of dopaminergic neurons in the substantia nigra was not performed. Dysfunction of the nigrostriatal system is the major cause of the observed symptoms in Parkinson’s disease. Therefore, this region is an important target for gene delivery aiming at disease modelling or gene therapy. In this study we compare LV and different rAAV serotypes on their transduction efficiency and cell specificity for dopaminergic neurons of the nigrostriatal pathway. Methods: LV were produced in 293T cells after a triple transient transfection with a transfer (CMV-eGFP-T2A-Fluc), packaging and envelope plasmid (VSV-G). Recombinant AAV vectors were generated in 293T cells using a CMVeGFP-T2A-Fluc expression cassette and packaged using capsid sequences from AAV 1, 2, 5, 6, 7 or 9. Each vector type was stereotactically injected into the mouse and rat substantia nigra. Transduction efficiency and distribution of the vector was determined by immunohistochemical analysis of eGFP expression in the substantia nigra and projections into the striatum. To determine the dopaminergic cell specificity, co-labelling with eGFP and tyrosine hydroxylase was performed. Further, we used in vivo bioluminescence imaging (BLI) to quantify and compare gene transfer into the rodent substantia nigra in a non-invasive way. Results and conclusion: The first results show that several rAAV serotypes are more efficient in transducing dopaminergic neurons compared to LV. Further detailed analysis will reveal the most promising rAAV serotype for gene delivery of dopaminergic neurons in the substantia nigra. We will discuss these data in relation with the outcome of the BLI data. These results demonstrate the use and effi-

ESGCT 2008 POSTER PRESENTATIONS ciency of rAAV for disease modeling and their potential for gene therapy in the nigrostriatal pathway. E-mail: [email protected] P 87 Effect of lentiviral vector expressing neurotrophin-3 and shRNA for NG2 on cellular environment of injured spinal cord Eleanor Donnelly 1; Siobhan McMahon 2; Peter Dockery 2; John Fraher 3; Padraig Strappe 4; Daniel O’Toole 5; Lisa McGinley 1; Timothy O’Brien 1 1NUI Galway, Regenerative Medicine Institute, Galway, Ireland; 2NUI Galway, Anatomy, Galway, Ireland; 3University College Cork, Anatomy, Cork, Ireland; 4University Sydney, Centre for Brain Research, Sydney, Australia; 5NUI Galway, Anesthesia, Galway, Ireland

A glial scar forms following injury to the spinal cord. This scar prevents regeneration and comprises mainly of chondroitin sulphate proteoglycans, such as NG2. In this study, short hairpin RNAs have been designed to target NG2 and have been cloned into a lentiviral vector. The Neu7 cell line is a rat astrocyte cell line which over expresses NG2, thereby mimicking the inhibitory atmosphere following spinal cord injury. The lentiviral vector containing the shRNA for NG2 was used to transduce Neu7 cells. RNA was extracted from these cells and following reverse transcription PCR, densitometry was performed on the resulting gel. 60% knockdown of NG2 was seen in virally-treated Neu7 cells compared to non-treated cells. A lentiviral vector was also constructed containing neurotrophin-3 (NT-3). Studies in our laboratory have shown that NT-3 can promote axon elongation. We are currently using the NT-3 vector in combination with the vector containing shRNA for NG2 to provide a dual approach to regenerating injured spinal cord, by knocking down the inhibitory atmosphere and delivering a neurotrophic factor to encourage axon growth across the site of injury. The functional effect both of these vectors was shown in a co-culture system with dorsal root ganglia (DRG) neurons to determine the effect on DRG neurite of knocking down NG2 and promoting NT-3 production within Neu7 cells outgrowth. Lentiviral vector expressing NT-3, shRNA to NG2 or a combination of both vectors were directly injected into contused rat spinal cord at T9 one week post injury. Animals were sacrificed two weeks post injection of virus and examined for changes in expression of NG2 and NT-3. This approach shows much promise as a therapeutic strategy for promoting nerve regeneration and reducing inhibitory factors within the lesion site of a spinal cord injury. E-mail: [email protected] P 88 Non-viral gene therapy using a C-terminal fragment of tetanus toxin heavy chain encoding plasmid improves motor function, inhibits apoptotic pathways and prolongs survival in a mouse ALS model Maria Moreno-Igoa 1; Ana Cristina Calvo 1; Clara Pena 2; Raquel Manzano 1; Maria Jesus Muñoz 1; Pilar Zaragoza 1; Jose Aguilera 3; Xabier Navarro 2; Pinzolas Osta 1 1Universidad

de Zaragoza, Lagenbio-I3A, Zaragoza, Spain; Autònoma de Barcelona, Department of Cell Biology, Physiology and Immunol, Institute of Neurosciences, Barcelona, Spain; 3Universitat Autònoma de Barcelona, Department of Biochemistry and Molecular Biology, Institute of Neurosciences, Barcelona, Spain 2Universitat

ESGCT 2008 POSTER PRESENTATIONS Transgenic mice expressing mutant human superoxide dismutase with substitution of glycine to alanine in position 93 (SOD1G93A) constitute a model of familial amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease with no effective therapy known. Previous in vitro studies have described some signaling pathways underlying the administration of the atoxic C-terminal fragment of tetanus toxin heavy chain (TTC) to neurons. TTC has been reported to be implicated in the activation of cascades responsible for trophic actions and neuroprotection through anti-apoptotic effects. We investigated whether these properties may be maintained in the in vivo ALS model. Naked-DNA encoding for TTC was intramuscularly injected at the presymptomatic age of 8 weeks in four limbs, and neuromuscular function and clinical behaviour were monitored until endstage. Our results indicate that TTC treatment reduced the decline in hindlimb muscles innervation, significantly delayed onset of symptoms and functional deficits, improved spinal motoneuron survival, and prolonged lifespan by approximately 13%. Based on the retrograde transport ability of TTC, we examined the spinal cord of SOD1G93A mice at symptomatic age. Using real-time PCR we found that caspase-1 and caspase-3 proapoptotic genes were down-regulated in treated mice and the active form of caspase-3 was inhibited, as shown by western-blot analysis. Survival signals, such as phosphorilation of Akt, were also detected after TTC treatment. These results suggest that non-viral gene therapy using the TTC-encoding plasmid provides a potential therapy for ALS. This work was supported by the grant of CAJA NAVARRA: “Tú eliges, tu decides”. PI071133, PI060201 and funds of CIBERNED from the Fondo de Investigación Sanitaria of Spain. PROFIT funds from the Ministerio de Educación y Ciencia and Action COST-B30 of the EC. E-mail: [email protected] P 89 AAV-mediated sulphamidase expression in the liver prevents development of somatic alterations in MPS IIIA mice Albert Ruzo ; Miquel García ; Virginia Haurigot ; Antonella Chiandetti ; Xavier M. Anguela ; Carles Roca ; Xavier León ; Eduard Ayuso ; Jesús Ruberte ; Fatima Bosch Autonomous University of Barcelona, CBATEG, Barcelona, Spain Mucopolysaccharidosis IIIA (Sanfilippo syndrome type A) is a lysosomal storage disease caused by the lack of sulphamidase (Sgsh) activity which is needed for the complete degradation of heparan sulphate (HS) glycosaminoglycan. This results in widespread HS accumulation and causes both neurodegeneration and somatic pathology, leading to death around puberty. There is no effective therapy available. The aim of this study was to evaluate a new gene therapy approach for this disease based on the genetic manipulation of either the liver or the skeletal muscle of a MPSIIIA mouse model to secrete the lacking enzyme to the bloodstream. Circulating sulphamidase can be taken up by non-transduced cells via the mannose-6-phosphate receptor, where it is targeted to the lysosomes and can correct the HS accumulation. With this objective, we generated a serotype 8 adeno-associated viral vector which expressed the mouse sulphamidase cDNA (AAV2/8-Sgsh). MPSIIIA mice receiving an intravenous or an intramuscular administration of AAV2/8-Sgsh

1131 showed a high expression and activity of sulphamidase in liver or in muscle, respectively. These tissues were able to secrete the enzyme to the bloodstream, as the sulphamidase activity in the blood serum almost reached the levels of control mice, whereas it was undetectable in MPSIIIA untreated mice. Glycosaminoglycan (GAG) accumulation was decreased in all tissues, even reaching normalization in many of them, demonstrating that the enzyme was being taken up by non-transduced cells. Accordingly, the urinary excretion of GAGs was completely normalized. Furthermore, a significant reduction in the GAG accumulation was found in the brain of the treated animals. Our results suggest that a therapy for the MPS IIIA based on the genetic modification of the liver or the muscle could benefit both the peripheral tissues and also the central nervous system. Therefore, our study suggests that this therapeutic approach may be of great interest as it would improve the lifespan and the quality of life of MPSIIIA patients. E-mail: [email protected] P 90 3 trans-splicing repair of the COL7A1 gene in epidermolysis bullosa dystrophica patients Eva Maria Murauer 1; Alfred Klausegger 1; Lloyd G. Mitchell 2; Johann W. Bauer 1 1eb-house austria, Laboratory for Molecular Therapy, Salzburg, Austria; 2Retro Therapy, Laboratory for Molecular Therapy, Bethesda, United States

Background: Dystrophic EB (DEB) is an inherited skin disease, which results from mutations in the COL7A1 gene, encoding type VII collagen, the major structural component of anchoring fibrils. Until now gene-therapeutic approaches in DEB have been hindered by the size of the COL7A1 transcript (9 kb). Method: To overcome this problem the size of the therapeutic insertions can be reduced by spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level by replacing mutant exons. The applicability of SMaRT in skin gene therapy has been proven in a -galactosidase (-gal) double transfection trans-splicing assay in 293T cells. In this system we used intron 43 of the COL7A1 gene as the target for trans-splicing. Results: Expression of the -gal protein was observed only in cells cotransfected with a pre-trans-splicing molecule (PTM) consisting of a binding domain (BD) of intron 43. In a series of trans-splicing experiments using different PTMs for intron 43 and intron 64 we have noticed that an increase in efficiency can be obtained by varying the BD of the PTM. To determine the optimal binding sequences for the targeted intron of the COL7A1 gene, we are performing a high throughput screening method. Conclusion: The results of the -gal experiments confirmed that trans-splicing is accurate and functional and showed specific trans-splicing in 10 % of all transfected cells. In a double transfection assay in 293T cells using the highthroughput system we could even select single PTMs showing a 80 % trans-splicing efficiency. This in vitro pre-evaluation of the PTMs highly improves the selection of binding domains, which is up to now based on trial and error. The selected PTMs can be used for gene-correction in keratinocytes and fibroblast from COL7A1-deficient patients, which constitutes a novel approach to treat DEB patients. E-mail: [email protected]

1132

ESGCT 2008 POSTER PRESENTATIONS

P 91

patients suffer from severe and recurrent life-threatening infections. One strategy for the treatment of patients lacking a matched donor for bone marrow transplantation is a gene replacement therapy. The occurrence of a clonal expansion of gene-modified cells in the X-CGD trial and the severe adverse events in the X-SCID trials clearly demonstrate the requirement for new vectors with reduced genotoxicity. The use of SIN-lentiviral vectors with specific internal cellular promoters could be an alternative to the currently used vectors. We compared a series of SIN-lentiviral vectors containing internal myeloid specific promoters driving the expression of codon optimized gp91 in two initial ex-vivo experiments. In our first set of tests in PLB-XCGD cells we could show a pronounced increase in the expression of gp91 upon DMSO-induced granulocytic differentiation to levels comparable to wild-type PLBs. In FACS-sorted populations of cells transduced at low MOI the vector copy number (VCN) was close to one, indicating that each integrated vector expresses the transgene. Further experiments in murine lineage-negative cells isolated from X-CGD mice revealed that expression of gp91 from our specific vectors is virtually absent in KSL-cells and that they mediate strong expression of gp91 when these cells are differentiated towards the granulocytic lineage. In this experiment we could also demonstrate rescue of the CGD-phenotype which correlates with the fraction of transduced cells as shown by DHR-assay. The most promising candidate was tested in-vivo in mice. Expression of gp91 from this vector was limited to differentiated myeloid cells with a peak expression in mature granulocytes. After 16weeks the mice were sacrificed for final analysis and expression of gp91 in bone marrow and spleen was detected by FACS. Furthermore we could show that expression of gp91 in bone marrow KSL cells was absent. Further molecular analysis to determine the levels of engraftment and vector copy number of engrafted cells are ongoing.

Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted retroviral vectors Giulietta Maruggi 1; Francesca Di Nunzio 1; Alessia Cavazza 1; Stefano Ferrari 2; Enzo Di Iorio 2; Marta Garcia 3; Michele De Luca 2; Graziella Pellegrini 2; Marcela Del Rio 3; Fernando Larcher 3; Fulvio Mavilio 1 1Università

di Modena, Scienze Biomediche, Modena, Italy; Eye Bank Foundation, Veneto Eye Bank Foundation, Venice, Italy; 3CIEMAT-CIBERER U714, Epithelial Biomedicine Division, Madrid, Spain 2Veneto

Mutations in any of the genes encoding the laminin-5 (LAM5) heterotrimer cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. In a pilot clinical trial, we recently showed that transplantation of cultured skin derived from autologous epidermal stem cells transduced with a MLV-derived retroviral vector expressing the beta3-chain cDNA reconstitutes normal synthesis and secretion of laminin-5, and corrects the adhesion defect in vitro and in vivo. Since the use of retroviral vectors carrying wildtype LTRs in human stem cells is considered unsafe due to the genotoxic risk associated to insertional gene activation, we developed an alternative gene transfer strategy based on self-inactivating (SIN) MLV-derived gamma-retroviral and HIV-derived lentiviral vectors in which expression of either GFP or a cDNA is under the control of the keratinocyte-specific, basal layer-restricted promoter-enhancer elements of the keratin 14 (K14) gene. Analysis of regenerated human skin upon grafting of bioengineered skin equivalents on immunodeficient mice showed that GFP expression directed by the K14 elements is tissue-specific and restricted to the basal layer of the epidermis. Correction of laminin 5 deficiency from the different vectors was evaluated in keratinocytes derived from skin biopsies of JEB patients in vitro and in vivo. These experiments demonstrated transduction of repopulating epidermal stem cells and full phenotypic correction of the JEB defect in a relevant pre-clinical model. The effect of the K14 and of the MLV LTR enhancer on the expression of genes surrounding the vector insertion sites was analyzed in clones of keratinocytes harboring 1 to 5 integrated proviruses by a quantitative assay. This study shows that the use of retroviral vectors carrying a tissue-specific, restricted promoter is an effective, and potentially safer, alternative for gene therapy of JEB. E-mail: [email protected] P 92 Lentiviral vectors for gene-therapy of X-CGD: specific and high expression of gp91phox restricted to granulocytes Christian Brendel 1; Stefan Stein 1; Michael Antoniou 2; Manuel Grez 1 1Institute

for Biomedical Research, Frankfurt, Germany; Biology Group, Medical and Molecular Genetics, Kings College London School of Medicine, London, United Kingdom 2Nuclear

Chronic Granulomatous Disease (CGD) is a rare inherited immunodeficiency caused by mutations in any of the subunits of the phagocytic NADPH oxidase. Due to the lack of NAPDH oxidase activity, neutrophils of CGD patients are unable to generate superoxide which is required for the killing of intracellular pathogens. As a consequence, CGD

E-mail: [email protected] P 93 Lentiviral vector development for X-linked hyper IgM syndrome (XHIGM-1) for gene therapy Zulema Romero 1; Juan Diego Unciti 1; Marien Cobo 2; Francisco Martin 2; Ignacio Molina 1 1Biopatogy and Regenarative Medicine Institute, Immunology, Granada, Spain; 2Institute of Parasitology and Biomedicine López Ne, Immunology and Cell Biology Department, Grnanada, Spain

Background: XHGM-1 is an X-linked immunodeficiency characterized by failure in immunoglobulin isotype switching caused by mutations in CD40L gene expressed in CD4 T cells. Autologous HSCs gene replacement is envisioned as a promising alternative therapy. However, CD40L gene is tightly regulated and GT approaches using constitutive vectors induced laeukemia in animal models. Therefore, we propose the development of lentiviral vector physiologically regulated to get a tight regulation of CD40L. Methods: We developed transcriptionally regulated lentiviral vector expressing CD40L. We used WASp-promoter to achieve haematopoietic specificity and SFFV promoter as control for constitutive expression (WCD40L and SCD40L respectively). WCD40L- and SCD40L-transducedhaematopoietic and non-haematopoietic cells were analyzed for CD40L expression by FACS. Triter and vector copy number per cell were determined by q-PCR.

ESGCT 2008 POSTER PRESENTATIONS Results: We achieved tight control CD40L expression by WASp-prooter-driven Lentiviral vectors (WCD40L). WCD40L-transduced non-haematopoietic cells (MOI  1-2) were unable to express CD40L but achieve moderate protein levels in haematopoietic cells. However SCD40L achieve high level of protein in all cells analyzed. Conclusions: The moderate and haematopoietic-specific expression of CD40L by WCD40L could render a safer profile for XHGM-1 gene therapy approaches. However we are also working in the development of CD40L-expressing vectors driven by the CD40L promoter. E-mail: [email protected] P 94 WASP intragenic sequences enhance haematopoietic specificity of WASp-promoter-driven lentiviral vectors Miguel Toscano 1; Pilar Muñoz 2; Karim Benabdellah 3; Cecilia Frecha 4; Marien Cobo 2; Francisco Martin 2 1Temple University School of Medicine, Department of Microbiology and Immunology, Philadelphia, United States; 2IPBLN, CSIC, Immunology and Cell Biology Department, Armilla, Granada, Spain; 3Estación Experimental del ZaidínCSIC, Microbiology, Granada, Spain; 4Université de Lyon, Human Virology Department, Lyon, France

Background: The development of haematopoietic-specific vectors is an important goal for gene therapy of haematological disorders. We have previously shown that a 500 bp fragment from the proximal WASp gene promoter in a lentiviral vector was sufficient to achieve over 100 higher levels of WASP protein in HCs than in non-haematopoietic cells. Methods: Haematopoietic and non-haematopietic cells were transduced with lentiviral vectors harbouring eGFP, eGFP-WASP chimera or eGFP and different WASP cDNA fragments. FACS, quantitative Western Blot and q-PCR were used to determine expression levels and vector copy number of transduced cells. Results: We demonstrated that the WASP cDNA play an important role in achieving highly specific haematopoietic expression. The effect of WASP cDNA is due to an effect on transcriptional activity on the WASp promoter and this effect is governed by a 5 900bp region of the WASP cDNA. We also showed that the WASP cDNA interferes with the enhancer activity of WPRE restricting its use to enhance transgene expression in our system. Conclusions: These results represent the first data showing the role of WASP intragenic sequences in WASp promoter activity that can be used for the construction of more haematopoietic-specific lentiviral vectors. E-mail: [email protected] P 95 Lentiviral transfer of the human IL2R gene into lineage depleted hematopoietic cells results in efficient phenotypic correction of IL2R/ mice Marshall Huston 1; Niek van Til 1; Truidi Visser 1; Karin Pike-Overzet 2; Frank Staal 2; Gerard Wagemaker 1 1Erasmus MC, Hematology, Rotterdam, Netherlands; 2Erasmus MC, Immunology, Rotterdam, Netherlands

Gene therapy for X-linked severe combined immunodeficiency (X-SCID) by gammaretroviral vectors to deliver a functional copy of the IL2RG gene into the host genome has

1133 been very effective in the clinical setting, but carries the risk of insertional oncogenesis (Hacein-Bey-Abina, Science 2003). HIV-1 derived lentiviral vectors have advantages over gammaretroviruses in transducing quiescent cells, such as longterm repopulating hematopoietic stem cells (HSC), are less likely to integrate near transcription start sites, and are thought to have lower genotoxicity (Montini, Nat Biotechnol 2006). To test efficacy and safety of lentiviral IL2RG gene therapy for X-SCID, a self-inactivating lentiviral vector was constructed containing a sequence-optimized human IL2RG gene (IL2RGopt). Four different promoters were tested: SFFV, EFS, PGK and the native human IL2RG promoter. Lineage negative HSC of IL2RG / mice were transduced with these vectors or a GFP control vector, resulting in 1 to 5 transgene copies per transduced cell with above 80% efficiency. The transduced cells were transplanted into 6 Gy irradiated IL2RG / mice and compared to IL2RG / recipients of wild type BALB/c cells. Blood collected monthly and analyzed by differential cell counting and immunophenotyping demonstrated that mice transplanted with cells transduced with the IL2RGopt transgene reconstituted T cells indistinguishable from those transplanted with healthy BALB/c cells, whereas recipients of cells transduced with GFP failed. B cell levels varied depending on the promoter but in all cases were present at levels at least 100-fold higher than GFP controls. IL-2/Con-A spleen cell proliferation assays and IgM/IgG1 serum levels in treated mice yielded results similar to wild type controls. Full functional immune analyses is in progress, as is monitoring of potential adverse effects. We conclude that lentiviral-based IL2RGopt gene therapy is an effective alternative to gammaretroviral gene delivery and that the PGK promoter and native IL2RG promoter region are efficacious candidates for future gene therapy trials. Current experiments aim at establishing the minimum cell dose required for efficacy without conditioning of the recipients, integration analyses and long-term follow-up. E-mail: [email protected] P 96 Development of lentiviral vectors for haemophilia a gene therapy Christelle J.M. Sion 1; Janka Matrai 2; Marinee K. Chuah 2; Thierry VandenDriessche 2; Susan M. Kingsman 1; Kyriacos A. Mitrophanous 1; Pippa A. Radcliffe 1 1Oxford

BioMedica, Virology, Oxford, United Kingdom; institute for biotechnology, VIB, Leuven, Belgium

2Flanders

Minimal lentiviral vectors have promise for gene therapy approaches for haemophilia A. These vectors have been engineered to lack most or all viral accessory genes and only a small proportion of the original viral genome is integrated into target cells. Plasma levels of factor VIII (FVIII) as low as 1-2 % of normal are of clinical benefit. However, FVIII is poorly expressed and in gene therapy clinical trials to date, sustained expression of FVIII at therapeutic levels has not been achieved. With the aim of maximising gene transfer in vivo, vectors encoding a secreted reporter gene (secreted alkaline phosphatase) were pseudotyped with a range of envelopes and administered intravenously to mice. Of the five envelopes tested VSV-G, baculovirus GP64 and Ebola envelopes resulted in stable expression for the three month duration of the study with the VSV-G pseudotyped vector conferring highest expression. We have previously shown that FVIII expression in producer cells inhibits vector production by in-

1134 hibition of pseudotyping 1 . For all envelopes tested this resulted in a reduction in functional titre. To circumvent this, FVIII expression was reduced in producer cells by use of a liver specific promoter and titres were restored. In vivo testing of different liver specific promoters is currently ongoing in order to maximise expression. We have also demonstrated that codon-optimisation of B domain deleted (BDD) FVIII improves expression both in vitro1 and in vivo. In conclusion, the vector of choice encodes codon-optimised BDD FVIII under the control of a liver specific promoter and is pseudotyped with VSV-G. 1 Radcliffe PA, Sion CJ, Wilkes FJ, Custard EJ, Beard GL, Kingsman SM, Mitrophanous KA. Analysis of factor VIII mediated suppression of lentiviral vector titres. Gene Ther. 2008 Feb;15(4):289-97. Epub 2007 Nov 29. E-mail: [email protected] P 97 Novel lentiviral vectors for haemophilia gene therapy Vincent Kao 1; Simon Waddington 2; Adrian Thrasher 3; Michael Antoniou 1 1King’s

College London, Medical and Molecular Genetics, London, United Kingdom; 2University College London, Haemophilia Centre and Haemostasis Unit, London, United Kingdom; 3University College London, Institute of Child Health, London, United Kingdom

Novel lentiviral vectors (LV) have been designed for efficient expression in the haematopoietic system for haemophilia gene therapy. The first LV consists of an expression cassette based on the erythroid specific human globin gene (locus control region elements, promoter, second intron/polyadenylation site) augmented with either (i) the GATA-1 gene HS2 insulator element (GATA-1/HS2) inserted within 3 LTR of the LV backbone or (ii) the ubiquitous chromatin opening element from the human CBX3/HNRPA2B1 locus (A2UCOE) linked upstream of the -LCR. All LV are tested in erythroid K562 and MEL and non-erythroid cell lines initially harbouring an eGFP reporter gene. Human Factor IX cDNA (hF.IX) will then be incorporated into what is found to be the most efficacious expression cassette for ex vivo haematopoietic stem cell and in utero delivery gene therapy in haemophilia B mouse model systems. The second LV consists of an A2UCOE-hF.IX unit that can provide expression in all haematopoietic lineages. LVs will be assessed in ex vivo bone marrow and in utero test delivery systems in haemophilia B mice. E-mail: [email protected] P 98 Development of foamy virus vectors for the genetic correction of chronic granulomatous disease Ilenia Chatziandreou 1; Elena. K Siapati 1; George Vassilopoulos 2 1Biomedical Research Foundation Academy of Athens, Cell and Gene Therapy lab, Athens, Greece; 2University of Thessaly, Division of Haematology, Medical School, Larissa, Greece

Background: Chronic granulomatous disease (CGD) is a rare genetic disorder that affects phagocytosis and is due to the defective generation of NADPH oxidase. Patients suffer from recurrent and often life-threatening infections. The most common defects are caused by mutations on the X-

ESGCT 2008 POSTER PRESENTATIONS linked CYBB gene encoding the gp91phox subunit of flavocytochrome b558. The aim of this project is to develop vectors based on foamy virus (FV) in order to transfer a functional copy of the gp91phox gene and correct the CGD phenotype. Methods:. A codon optimised human gp91phox cDNA was cloned into a deleted FV backbone and cDNA expression was driven off the MscvLTR or the Pgk promoters. The human myeloid PLB 985-XCGD cells were used for assaying phenotypic correction. gp91phox mRNA levels was determined by RT-PCR and protein expression was determined by anti-gp91phox antibody staining and flow cytometry. Functional correction was determined by the NBT assay. Results: PLB-985 X-CGD cells were transduced with FV.Pgk.gp91phox vector and analysis by Flow cytometry showed 4% gp91phox positive cells with expression levels (as determined by MFI) reaching 90% of the wt cells. The gp91phox expressing cells were functionally corrected, scoring positive for the NBT test. The viral copy number per cell was 1.7 as determined by Real time PCR. Lin – cells from XCGD mice were transduced in vitro and showed efficient gene transfer rates with an MOI of 5. Methylcellulose colonies stained positive for the NBT test. Transduced cells have been transplanted in the X-CGD mouse model using a clinically relevant protocol and analysis is pending. Further studies with human CD34 X-CGD cells are ongoing. Conclusion: These results show: 1) production of high titer and stable FV-gp91phox vectors, 2) adequate levels of gp91phox for the functional correction of PLB-985 XCGD cells, 3) expression of the protein in primary haematopoietic cells. E-mail: [email protected] P 99 TCR gene transfer: MHC I and II-restricted MAGE epitopes as melanoma-specific immune targets Trudy Straetemans 1; Marieke Broertjes 1; Joost Drexhage 1; Miriam Coccoris 1; Bart Lambrecht 2; Pierre van der Bruggen 3; Pierre G. Coulie 3; Jan Willem Gratama 4; Ralph Willemsen 1; Reno Debets 1 1ErasmusMC-Daniel

den Hoed Cancer Centre, Laboratory of Experimental Tumor Immunology, Dept Medical Oncology, Rotterdam, Netherlands; 2ErasmusMC, Dept Pulmonary Diseases, Rotterdam, Netherlands; 3Universite Catholique de Louvain, Ludwig Institute for Cancer Research, and Cellular Genetics, Brussels, Belgium; 4ErasmusMC-Daniel den Hoed Cancer Centre, Clinical Tumor Immunology, Dept Medical Oncology, Rotterdam, Netherlands The present study aims to direct T cell therapy of melanoma towards the MAGE-C2 and MAGE-A3 antigens, which are presented by HLA-A2 and HLA-DP4, respectively. These MAGE antigens are expressed by the majority of melanomas but not by normal adult tissues (and therefore considered safe target antigens) and have been demonstrated to induce tumor-specific and robust T cell responses in clinically responding patients. In our planned clinical trial, we anticipate to establish and maintain long-term anti-tumor activity by co-administration of MAGE-A3/HLA-DP4 (MA3/DP4) specific T cells (CD4 T helper cells) and MAGE-C2/HLA-A2 (MC2/A2) specific T cells (CD8 cytotoxic T cells). We cloned and characterized the MC2/A2 and MA3/DP4 TCR genes of CD8 and CD4 T cell clones derived from two metastatic melanoma patients who responded clinically to MAGE-vaccination, and subsequently retargeted human

ESGCT 2008 POSTER PRESENTATIONS T cells by TCR gene transfer. MC2 redirected CD8 T cells showed antigen-specific cytotoxicity and cytokine production upon co-incubation with a MC2-positive tumor cell line. MA3 redirected CD4 T cells showed antigen-specific proliferation and cytokine production upon co-incubation with autologous and mature, MA3-presenting dendritic cells. The in vivo efficacy and safety of MAGE-directed TCR gene therapy is tested in human A2 and DP4 transgenic mice transplanted with mouse melanoma cells expressing MC2/A2 and/or MA3/DP4. Receptor-engrafted T cells are currently tested for their effect on tumor growth, homing and peripheral persistence. Immunological side effects will be assessed by long-term follow up and general histopathology. To extend the concept of MAGE TCR gene therapy to non-melanoma tumors, we are currently determining the in vitro activity of MC2 and MA3 redirected T cells against a large panel of MAGE expressing human tumors such as mammary, easophagus and non-small cell lung cancers. Funding: Erasmus MC Translational Research and EU FW6 ATTACK E-mail: [email protected] P 100 Gene editing of naive and central memory T lymphocyte specificities for adoptive immune therapy of leukemia Elena Provasi 1; Oscar Muniz Pello 2; Zulma Magnani 1; Jurgen Kuball 3; Angelo Lombardo 2; Attilio Bondanza 1; Philip D. Gregory 4; Claudio Bordignon 5; Michael C. Holmes 6; Phil D. Greenberg 3; Luigi Naldini 2; Chiara Bonini 1 1San Raffaele Scientific Institute, Cancer Immunotherapy and Gene Therapy Program, Milan, Italy; 2San Raffaele Scientific Institute, Telethon Institute for Gene Therapy, Milan, Italy; 3Fred Hutchinson Cancer Research Center, Program in Immunology, Seattle, United States; 4Sangamo BioSciences, Research, Richmond, United States; 5San Raffaele Scientific Institute, San Raffaele University, Milan, Italy; 6Sangamo Biosciences, Therapeutic Gene Modification, Richmond, United States

Transfer of a T cell receptor (TCR) from a high-avidity tumor-specific T cell (CTLs) to polyclonal T cells may overcome the difficulty of expanding rare tumor-specific CTLs, under conditions that can preserve function and prevent exhaustion. Unfortunately, TCR-gene transfer is limited by: 1. Low and transient transgene expression; 2. Inappropriate pairing of the exogenous and endogenous TCR chains; 3. Poor survival and expansion of gene-modified effectors. We cloned genes encoding a high-avidity TCR specific for an HLA-A2-restricted peptide from the Wilms tumor antigen 1 (WT1), into a third generation lentiviral vector (LV) under the control of a bi-directional PGK or EF1a promoter. To increase TCR expression and facilitate appropriate TCR pairing, we used a codonoptimized TCR, modified with additional cysteines in the constant region of alpha and beta chains. T lymphocytes were efficiently transduced by both vectors following activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/ CD28) and culture with low doses of IL-7/IL-15. However, the PGK promoter was superior to EF1a in sustaining stochiometric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 50 days after stimulation. We observed that early T cell differentiation phenotype (CD45RA /CD62L, CD28 CD27, IL7Ra, IL-2 gIFN /), was preserved in TCRmodified lymphocytes. Sorted naive (CD45RA/CD62L) and central memory (CD45RA /CD62L) lymphocytes were

1135 efficiently transduced by TCR-LV and maintained the original phenotype. Upon antigenic stimulation, TCR-modified lymphocytes mediated WT1-specific gIFN production and cytotoxicity. To further improve the safety of the strategy, we attempted site-specific integration of transgenes by using of designed zinc finger nucleases (ZFN). Adenoviral transfer of a set of ZFN specific for CCR5 locus coupled with integrase defective lentiviral vectors carrying transgenes, enabled efficient site-specific integration in human lymphocytes resulting in stable transgene expression. Site-specific integration of optimized, leukemia-specific TCR into naive and central memory lymphocytes may represent an effective method for the generation of robust engineered tumor-specific CTLs. E-mail: [email protected] P 101 In vivo HIF-1 expression in experimental murine atherosclerosis Jeremy Ben-Shoshan 1; Arnon Afek 2; Sofia MayselAuslender 1; Adi Mor 1; Gad Keren 1; Jacob George

1

1Sourasky

Tel-Aviv Medical Center, Cardiology, Tel-Aviv, Israel; 2Sheba Medical Center, Pathology, Tel-Hashomer, Israel

Background: The T cell-mediated inflammatory balance has been well established as a key player in the progression of atherosclerosis. Recently, hypoxia and its principal molecular signature hypoxia inducible factor-1 (HIF-1) have been suggested to tune down inflammation by dictating anti-inflammatory programs. Here, we describe an inhibitory effect of HIF1 on atherosclerosis using a model of hydrodynamic gene injection in ApoE knockout mice. We also suggest a mechanism, which demonstrate for the first time a direct impact of HIF-1 on naturally occurring CD4CD25Foxp3 Tregs. Methods: Firstly, ApoE / mice were submitted to intravenous hydrodynamic injection of an empty vector or HIF-1 stable form, HIF-1P564A. After 24hrs, HIF-1 expression in splenocytes (n  3) was validated by RT-PCR and ELISA. One week post-injection, spleens and aortas (n  3) were analyzed for expression of IL-10, INF- and TGF- by RT-PCR. Protein array of harvested splenocytes was used to evaluate cytokine expression. Analysis of the number and suppression capacity of regulatory T cells sorted from the harvested splenocytes was performed by CD4CD25 Foxp3 triple staining flow cytometry and [3H]thymidine assay, respectively. At day 30, systemic injection (n  8) was repeated. At day 60, animals were scarified and aortas were isolated for plaque assessment. Results: Increased expressions of IL-10 and TGF- in splenocytes as well as a decreased expression of INF- in aortas were measured after one week in HIF-1 treated mice. Protein array of harvested splenocytes revealed a shift of the TH1 inflammatory response toward TH2 response. Increased number and suppression capacity of CD4CD25Foxp3 Tregs isolated from the harvested splenocytes was found in mice injected with HIF-1. At end point, aortic sinus lesion size was significantly decreased in mice treated with HIF-1 with reduced lipid cores and reduced INF- expression, compared with controls. Conclusion: Constitutive expression of HIF-1 in lymphocytes induces an inflammatory shifting from TH1 toward TH2 response, potentially by increasing the number and suppressive capacities of CD4CD25Foxp3 Tregs. These properties might be effectively employed to attenuate atherosclerosis progression. E-mail: [email protected]

1136

ESGCT 2008 POSTER PRESENTATIONS

P 102 Generation of a new chimeric antigen receptor (CAR) to target CD23 expressed on B-chronic lymphocytic leukemia (B-CLL) cells Greta Maria Paola Giordano Attianese 1; Vera Hoyos 2; Barbara Savoldo 2; Virna Marin 1; Malika Brandi 1; Andrea Biondi 1; Ettore Biagi 1; GianPietro Dotti 2 1M.Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca, Monza, Italy; 2Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, United States

Background: B-Chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of mature Blymphocytes expressing CD19, CD20dim and aberrantly expressing the CD5 T-cell marker. Moreover, they over-express the B-cell activation marker CD23. Chimeric Antigen Receptors (CAR) are engineered molecules able to redirect Tcell killing/effector activity towards a selected target in a non MHC-restricted manner. First trials targeting B-CLL were based on both monoclonal antibodies and antiCD19/anti-CD20 CAR-transduced T cells. However, this approach causes the elimination of normal B-lymphocytes and B-precursors with consequent impairment of humoral responses. Selective CD23 expression on B-CLL cells renders it an optimal target to design a specific CAR. Methods: a new CD23-targeting CAR to redirect T cells against CD23 B-CLL has been generated. After transduction, modified T cells, were tested for cytotoxicty against different CD23-targets using a classic chromium release assay. Results: The anti-CD23 CAR was stably expressed by primary T cells after transduction (average expression, 20%; range, 10%-60%; n  10) and conferred them a strong cytotoxicity against CD23 tumor cell lines (Epstein Barr Virus transformed lymphoblastoid cell line, average lysis, 50%; range 15%-70%, at Effector:Target Ratio (E:T)  40:1; n  5; Bjab and Jeko cell lines transduced with human CD23 antigen, average lysis, 60%; range, 20%-75%, at E:T  40:1; n  3). On the contrary, anti-CD23 transduced T-cells displayed no relevant killing versus normal B cells (average lysis, 8%; range, 1%-15% at E:T ratio 40:1; n  3), differently from antiCD19 CAR redirected T-cells, which killed tumor and normal B cells in an indistinct manner. Preliminary results indicated that T cells from B-CLL patients can also be efficiently transduced with the anti-CD23 CAR (average expression, 80%; range, 70%-90%; n  3) and redirected specifically toward autologous blasts (average lysis, 29%; range, 21%-35% at E:T  20:1; n  3), without being inhibited by soluble CD23-enriched autologous plasma. Conclusion: CD23-targeting through a specific CAR, for the potentiality to get selective and potent killing of tumor cells, while sparing normal B cells, holds great promises for adoptive immunotherapy of B-CLL. E-mail: [email protected]

Resistance of mature T cells to oncogene transformation 2;

3;

Sebastian Newrzela Kerstin Cornils Zhixiong Li Christopher Baum 3; Johann Meyer 3; Martijn H. Brugman 3; Marianne Hartmann 1; Nabil Al-Ghaili 1; Julia Schlaeger 1; Ji-Hee Yi 1; Sylvia Hartmann 4; Martin-Leo Hansmann 4; Boris Fehse 2; Dorothee von Laer 1 1Georg-Speyer-Haus,

Leukemia caused by retroviral insertional mutagenesis after stem-cell gene transfer has been reported in several experimental animals and in patients treated for X-linked SCID. We analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental ‘worst case scenario’, we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gammaretroviral vectors encoding the potent T cell oncogenes LMO2, TCL1 or TrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1 deficient recipients (Ly5.2), stem cell transplanted animals developed T cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated PCR analysis revealed mono- or oligoclonality of the malignancies. In striking contrast, none of the mice transplanted with T cells transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. In vitro, an immortalized T cell clone was generated after transduction with LMO2. This clone was CD4/CD8 double negative but showed a recombined T cell receptor. In vitro the clone overgrew a non-manipulated competitor T cell population, but in vivo no tumor formation was observed after transplantation in Rag-1 recipients. Further analysis revealed a very interesting retroviral integration site near the genes for the alpha chains of the IL-2 and IL-15 receptors. Both receptors were constitutively up regulated in this T cell clone. Ectopic expressions of both receptor chains alone, in combination and in combination with LMO2 were performed, to analyse the possible role of these gene products in the immortalization process. This clone could be the first example, were insertional mutagenesis has contributed to the immortalization of a mature T cell clone. In summary our results indicate that mature T cell populations are less prone to transformation by known T cell oncogenes, but that certain conditions indeed may support such an event. Investigations addressing the mechanisms of the relative resistance of mature T cells to oncogene transformation are currently under way. E-mail: [email protected] P 104 Adoptive transfer of murine T cells leads to rapid regression of human melanoma in SCID mice Miriam Coccoris ; Trudy Straetemans ; Cor Berrevoets ; Csilla Scholten ; Reno Debets ErasmusMC-Daniel den Hoed cancer center, Medical Oncology, Rotterdam, Netherlands

P 103 1;

Experimental Pediatric Oncology and Hematology, Frankfurt am Main, Germany; 3Hannover Medical School, Depart. of Hematology, Hemostaseology and Oncology, Hannover, Germany; 4University Hospital of the Goethe-University, Department of Pathology, Frankfurt am Main, Germany

AG von Laer, Frankfurt am Main, Germany; 2University Hospital of the Goethe-University,

Adoptive transfer (A.T.) of T cells redirected against a tumor antigen through TCR gene transfer is a promising approach to target tumors. In a recent phase I clinical trial it has been demonstrated that A.T. of TCR transduced T cells is feasible. However, the anti-melanoma response rate was only 12% and considered low as compared to the response rate observed after A.T. of tumor infiltrating T cells (51%). In analogy to studies with T cells engineered with antibody-based receptors, we have set up a murine model to test

ESGCT 2008 POSTER PRESENTATIONS the efficacy and safety of TCR gene therapy directed against a human tumor. SCID mice bearing HLA-A2, gp100 melanoma cells (i.e. FM3 cells) were adoptively transferred with both human and murine T cells transduced with a TCR directed against human gp100280-288/HLA-A2. We found that human T cells did not induce regression of the tumor whereas the murine cells did. Murine T cells, that co-expressed GFP next to the transgenic TCR, were found in blood, spleen, lymph nodes and the tumor at the time of tumor regression. Next to tumor regression, we observed autoimmune-like reactions in the mice, such as diarrhea and skin rash. Unexpectedly, we observed that mock-transduced murine T cells also induced tumor regression, which was equally strong as for the TCR modified T cells. T cell expansion and side effects were similar to those observed in the TCR group. These findings suggest that the use of murine T cells in combination with certain human xenograft tumors in SCID mice, such as melanoma (this study) but not previously reported breast or colon tumors, leads to xeno-reactivity as well as auto-reactivity of the transferred T cells. We consider our findings valuable to the field of preclinical therapy with engineered T cells, and are currently investigating the clonality of the observed T cell responses and attempting to confirm our findings with a second human melanoma. Funding: EU FP6 ATTACK E-mail: [email protected] P 105 TCR gene therapy: Minimal domains of CD3 transplanted onto TCR to induce highly preferential pairing between human TCR- and -chain Coen Govers ; Zsolt Sebestyen ; Ralph Willemsen ; Reno Debets Erasmus MC-Daniel den Hoed Cancer Center, Dept. Medical Oncology, Laboratory of Experimental Tumor Immunology, Rotterdam, Netherlands T cell receptor (TCR) gene therapy is hampered by diluted therapeutic TCR expression and generation of potentially self reactive T cells, both a direct consequence of TCR mispairing. We have shown previously that TCRs fused to complete human CD3 (TCR:CD3) prevents mispairing with endogenous TCR chains and induces highly preferential and CD3-independent pairing of TCR and  transgenes (Sebestyén, 2008). In this study, we set out to identify minimal domains of CD3 and TCR necessary to preserve preferential TCR pairing and at the same time regain the TCR’s ability to complex with endogenous CD3 (and to function in a TCR-like manner). We have employed a gene exchange strategy and generated minimal TCR:CD3 constructs, in which either the extracellular (EC), transmembrane (TM) or intracellular (IC) domains of CD3 or combinations thereof are replaced by the corresponding TCR domains. Dual TCR Jurkat T cells were made which expressed, next to MelanA/HLA-A2 TCR, MAGE-A1/HLA-A1 specific TCR that has one of the following formats: minimal TCR:CD3, complete TCR:CD3, non-modified TCR, or an alternatively modified CD3-dependent TCR containing murine constant domains and additional cysteine residues (mucys TCR). TCR-transduced Jurkat T cells were analyzed by flow cytometry for surface expression of the exogenous and endogenous TCRs and their binding to pMHC as well as the transgenic TCR’s ability to associate with endogenous CD3. TCR:CD3 demonstrated the highest expression per cell

1137 (MFI) and binding to pMHC of all TCR modifications tested, including mucys TCR. Minimal TCR:CD3 constructs containing TM CD3, but not the one without TM CD3, expressed at the surface of T cells and provided pMHC binding, albeit at lower levels than observed for complete TCR:CD3, and remained non-competitive for endogenous CD3. Studies to further fine-tune minimal TCR;CD3 constructs in an effort to enhance surface expression and at the same time reintroduce CD3 assembly and TCR/CD3 signaling are ongoing. For reasons of efficacy and safety, we consider the TCR:CD3 format applicable for clinical TCR gene therapy. Funding: This study is financially supported by FW6 ATTACK. E-mail: [email protected]

P 106 T cell receptor gene-modified T cells with specificity against a broadly expressed renal cell carcinoma antigen Matthias Leisegang 1; Adriana Turqueti 2; Thomas Blankenstein 3; Dolores Schendel 2; Elfriede Nößner 2; Wolfgang Uckert 1 1MDC,

Cell Biology and Gene Therapy, Berlin, Germany; Research Center for Environmental Health, Molecular Immunology, Munich, Germany; 3MDC, Tumor Immunology and Gene Therapy, Berlin, Germany 2German

T cells with reactivity to antigens expressed by tumor cells are found in solid tumors like renal cell carcinomas (RCC). However, tumor-infiltrating lymphocytes (TIL) are mostly not sufficient to control tumor growth. To boost the anti-tumor response, adoptive transfer of T cells genetically engineered by T cell receptor (TCR) gene transfer to achieve a new antigen specificity is one option to overcome the paucity of RCC-reactive T cells. We isolated TIL from a RCC patient. One TIL clone (TIL53) showed ex vivo cytotoxicity against several HLA-A2.1positive tumor cell lines without recognizing normal kidney cells. From this clone, a TCR (TCR-53) was molecularly cloned. The TCR-53 genes were used to generate an indicator cell line to screen RCC and other tumor lines and normal cells for their antigen distribution. For this purpose, mouse B3Z cells were modified by retroviral gene transfer of both, the TCR-53 genes and human CD8alpha. Upon activation by MHC-peptide binding, B3Z cells express beta-galactosidase and interleukin-2. 67% of HLA-A2.1-positive RCC lines and some cell lines of other tumor origin were recognized by TCR-53 expressing B3Z cells indicating the broad distribution of this RCC antigen whereas no reactivity to normal cell lines was found. Retroviral transfer of TCR-53 genes was employed to redirect the antigen specificity of primary human T cells. While wild-type TCR-53 gene-modified T cells were of low reactivity, T cells genetically engineered with codonoptimized and murinized TCR-53 genes showed an improved antigen recognition. These TCR engineered T cells exhibited cytotoxocity and cytokine response toward several tumor lines. In sum, we cloned a TCR from RCC-infiltrating TIL that allows the analysis of RCC antigen distribution in tumor and normal cells and the identification of the new tumor-associated antigen. TCR-53 modified T cells are an interesting tool for adoptive T cell therapy. E-mail: [email protected]

1138

ESGCT 2008 POSTER PRESENTATIONS

P 107

P 108

The coexpression of a partially murinized single chain TCR gp100 and a truncated TCR displays marked tumor antigen reactivity in vitro and in vivo

Investigation of the role of gene-modified CD4 T cell subsets for cancer immunotherapy

Ralf-Holger Voss 1; Simone Thomas 1; Christina Pfirschke 2; Beate Hauptrock 3; Margarete Grabowski 3; Ratna Sari Intan Poendl 3; Renate Engel 4; Philippe Guillaume 5; Pedro Romero 6; Christoph Huber 7; Philipp Beckhove 2; Matthias Theobald 8 1III.

Medical Clinic & Policlinic, Hematology & Oncology, Mainz, Germany; 2German Cancer Research Center (DKFZ), Translational Immunology Unit, Heidelberg, Germany; 3III.Medical Clinic, Hematology & Oncology, Mainz, Germany; 4European Institute for Research and Development, of Transplantation Strategies, Idar-Oberstein, Germany; 5Ludwig Institute for Cancer Research, Lausanne branch, Epalinges, Switzerland; 6Ludwig Institute for Cancer Research, Clinical Onco-Immunology, Lausanne, Switzerland; 7III.Medical Clinic & Policlinic, Hematology & Oncology, Mainz, Germany; 8University Medical Center, Hematology and Van Creveld Clinic, Utrecht, Netherlands Human TCR gene transfer therapy aims at introducing tumor-reactive TCRs into human T-cells for adoptive cell transfer therapy. However, the transfer of double chain (dc) TCRs into human T-cells carries the risk of hybrid TCR dimer formation with the endogenous TCRs potentially leading to autoimmunity. To prevent this, a 3-domain single chain (sc) TCR specific for the Melanoma-derived tumor antigen gp100 was constructed by connecting the variable Va-domain to TCRb. To provide the missing Ca-domain, a truncated TCRa consisting of the signal peptide/Ca domain was generated and coexpressed. After retroviral transduction of this construct into human T-cells, effector functions were strongly reduced when compared to T-cells transduced with a corresponding wild type TCR. Since murine sc TCR p53/Ca constructs are functional in human T-cells, the human Ca- and Cb-constant domains were replaced by the murine ones. The chimeric scTCR displayed good expression and function levels. However, the presence of murine domains may be immunogenic. To minimize this event, we identified subdomains or single amino acids responsible for the beneficial effect of partial murinization on functionality. The murinization of the constant ectodomains located N-terminally to the membrane proximal interchain disulfide bond yielded a substantial functionality as opposed to the murinization of the C-terminal subdomains. Subsequently, four amino acids in the human Cb ectodomain were replaced by the corresponding basic amino acids present in murine Cb. According to a TCR/CD3-complex model, two of them favourably interact with the CD3 complex whereas the other two may strengthen the interchain interaction of TCRa and -b. The introduction of the latter ones resulted in a moderate increase in cytokine secretion. Currently, we are determining further murine TCR structural elements that may contribute to effector function. T cells transduced with various TCR gp100 constructs were tested in NOD-SCID mice bearing a human melanoma cell line xenograft. After onset of tumor growth up to 5 million T cells were intravenously injected. In consecutive experiments tumor growth was markedly antagonized by both the wild type and partially murinized sc TCR-transgenic Tcells. E-mail: [email protected]

Phillip Darcy 1; Maria Moeller 1; Joseph Trapani 2; Mark Smyth 2; Michael Kershaw 1 1Peter

MacCallum Cancer Centre, Cancer Immunotherapy, Melbourne, Australia; 2Peter MacCallum Cancer Centre, Cancer Immunology Research, Melbourne, Australia Background: Gene-modified T cells have gained much interest in the immunotherapeutic treatment of cancer. In a recent study we have demonstrated significant improvement to this approach by combined transfer of antigen-specific gene-modified CD8 and CD4 T cells.1 However, given that specific subsets of CD4 T cells have been demonstrated to play key roles in anti-tumor rejection, we wanted to assess the contribution of either Th1 and Th2 CD4 T cell subtypes for redirected T cell immunotherapy. Methods: We have developed a novel method involving retroviral transduction and in vitro T cell polarization to generate gene-engineered mouse CD4 Th1 and Th2 cells or Th intermediate (Thi) cells expressing an anti-erbB2-CD28- chimeric receptor. Expression of the transgene in the different CD4 subsets was assesed by flow cytometry and antigen-specific in vitro function was determined by ELISA, thymidine incorporation and cytotoxicity assays. The ability of the various geneengineered T cell subsets to mediate tumor regression was determined following adoptive transfer into tumor-bearing mice and both flow cytometry and confocal microscopy were employed to assess persistence and localisation of transduced T cells at the tumor site. Results: Gene-modified Th1 and Th2 polarized CD4 cells were characterized by the preferential secretion of IFN- and IL-4, respectively. In adoptive transfer studies using an erbB2 lung metastasis model, complete survival of mice was observed when transduced Th1, Th2 or Thi CD4 cells were transferred in combination with an equivalent number of transduced CD8 T cells. Tumor rejection was consistently associated with transduced T cells at the tumor site and interleukin-2 secretion. However, the surviving mice treated with gene-modified Th1 CD4 cells were significantly more resistant to a subsequent challenge with a different erbB2 tumor (4T1.2) implanted subcutaneously. This result correlated with both increased expansion of Th1 CD4 and CD8 T cells in the blood and a greater number of these cells localizing to the tumor site following rechallenge. Conclusion: This data supports the use of gene-modified CD4 Th1 and CD8T cells for mediating a sustained antitumor response. 1Moeller et al., Blood. 2005;106:2995-3003. E-mail: [email protected] P 109 Comparison of inducible caspase 9, herpes simplex thymidine kinase, CD20 and mutant thymidylate kinase as suicide gene strategies for adoptive immunotherapy Virna Marin 1; Irene Pizzitola 1; Andrea Biondi 1; Ettore Biagi 1; Pule Martin 2 1M.Tettamanti

Research Center, Department of Pediatrics, University of Milano Bicocca, Monza, Italy; 2Department of Haematology, University College London, London, United Kingdom

Background: Genetic modification of T cells with transgenic native T-cell receptors (TCR) or chimeric antigen re-

ESGCT 2008 POSTER PRESENTATIONS ceptors (CAR) allows the generation of large numbers of Tcells against almost any cancer antigen.However, immunotherapy with TCR or CAR-modified T cells may result in toxicity related to direct target effects, unanticipated offtarget effects or possibly lymphoproliferation due to vector insertional mutagenesis.Therefore inclusion of a suicide gene in the viral vector is becoming increasingly necessary.Several suicide genes have been described: Herpes Simplex Virus Thymidine Kinase (HSVtk), human inducible Caspase 9 (iCasp9), human CD20 and a mutant human thymidylate kinase (mTMPK). We attempt to compare these strategies. Methods: Similar SFG-based bicistronic vectors were generated. Each suicide gene was cloned in frame with truncated CD34 marker gene (dCD34), separated by the Footand-Mouth Disease 2A peptide (2A) to allow 1:1 expression of suicide and marker gene. Transduced Jurkat T-cells were enriched by CD34-immunoselection and CD34-selected cells were tested for sensitivity to the corresponding activator in vitro. Results: iCasp9-2A-dCD34 transduced cells were rapidly killed by a chemical inducer of dimerizeration (CID), with 72% (61%-79%; n  3) of lysis after 24 hours, 85% of lysis (77%-90%; n  3) after 72h and 89% (86%-94%; n  3) of lysis after 7 days. Gancyclovir treated HSV-tk-2A-dCD34 expressing cells showed similar levels of killing but only after 3 days, (61%-87%; n  3) and CD20-2A-dCD34 and mTMPK2A-dCD34 transduced cells showed only minimal lysis at all time points analyzed (maximum lysis, 50% and 27% respectively after 7 days; n  3).The same results were obtained by analyzing apoptosis induction by drug treatment through Annexin-7AAD staining. After just 5 hours of incubation with CID, 65% of iCasp9-2A-dCD34 cells were apoptotic, whereas no effect was yet detectable for the other suicide genes. Conclusion: These results suggest that whilst iCasp9 and HSV-TK show comparable in vitro efficacy,the faster activity of iCasp9 might be advantageous in case of occurring severe toxicity and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy. E-mail: [email protected] P 110 Optimizing TCR gene transfer Annelies Jorritsma ; Raquel Gomez ; Moniek de Witte ; Bianca Heemskerk ; Ton Schumacher ; John Haanen The Netherlands Cancer Institute, Immunology, Amsterdam, Netherlands A recent phase I trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of tumorspecific T cell receptor (TCR) genes into autologous lymphocytes is feasible. Although durable engraftment of TCR-transduced cells was achieved in fifteen patients, only two of these patients demonstrated objective tumor regression of metastatic melanoma and the further improvement of TCR gene therapy will be a major point of focus in the coming years. We have developed a mouse model in order to determine the requirements for optimal tumor targeting. These experiments revealed three factors that contribute greatly to the in vivo potency of TCR modified T cells. First, irradiationinduced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR se-

1139 quences has a profound effect on in vivo anti-tumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Implementation of these factors in the design of clinical trials is likely to positively affect the clinical efficacy of TCR gene therapy. In addition, we hypothesized that the success rate of TCR gene therapy may well be enhanced by the use of melanomareactive TCRs with a higher capacity for tumor cell recognition. Such enhanced recognition may either be brought about by increasing the expression level of a melanoma-reactive TCR, or by selecting more avid melanoma-reactive TCRs. From a panel of human, melanoma-reactive TCRs we selected the TCR with the highest expression and affinity. Furthermore, we assessed the lack of alloreactivity of this TCR against a large collection of common human leukocyte antigen alleles. The TCR selected in this project appears markedly superior as compared to the TCR recently used in a phase I clinical trial and this TCR will be used in an upcoming clinical trial at the NKI-AVL. E-mail: [email protected] P 111 Re-programming T cells through lentiviral mediated FOXP3 expression Hong Zhan ; Nichola Cooper ; Hubert Gaspar ; Waseem Qasim Institute of Child Health, Molecular Immunology Unit, London, United Kingdom Background: Modulating T cell function is of interest in a wide range of immune mediated pathologies. Ectopic expression of FOXP3, the master transcription factor in regulatory T cells, can confer suppressive properties. Lentiviral delivery to primary T cells offers the possibility of generating large numbers of re-programmed cells with regulatory properties. Methods: Self inactivating HIV-1 based lentiviral vectors were generated to express human FOXP3, with expression linked to truncated low affinity growth factor receptor (LNGFR) to facilitate identification and selection of transduced cells. Vectors included a central polypurine tract element, WPRE and a strong retroviral promoter derived from the spleen focus forming virus.T cells were activated with CD3/CD28 beads, or IL2, or in mixed lymphocyte culture. Highly efficient transduction was achieved using unconcentrated vector pseudotyped with chimaeric RD114/MLV envelope, or concentrated vector pseudotyped with vesicular stomatitis virus envelope. Transduced cells were rested and their phenotype characterised. Their ability to suppress proliferation assessed on the basis of 3H thymidine uptake and the dilution of CFSE on flow cytometry. Results: Concordant expression of FOXP3 and the marker LNGFR was demonstrated, and magnetic bead selection allowed for isolation of highly enriched populations. We show that polyclonally transduced FOXP3 expressing T cells suppress the proliferation of anti-CD3 stimulated peripheral blood lymphocytes in a cell dose dependent manner. Similarly, transduced alloreactive T cells can be reprogrammed to exhibit regulatory properties. Conclusion: Lentiviral mediated FOXP3 expression confers regulatory properties, and this may allow the development of novel therapies to target T cell mediated autoimmune diseases and allo-immune responses in the transplant setting. E-mail: [email protected]

1140

ESGCT 2008 POSTER PRESENTATIONS

P 112

modified T cells is limited, requiring repeated treatments due to AICD (activation induced cell death) upon exposure of these T cells to antigen. This limits the ability of the therapy to be self-sustaining and will limit the efficacy of the approach against larger tumors. To overcome the problem of reduced activation and survival of the first generation T cells, we created a “3rd generation” (G3), tripartite CIR against EGFRvIII (MRI-CD8TM-CD28-OX40-CD3), which contains both co-stimulatory factors CD28 and OX40. To investigate cell growth of G1 or G3 T cells via antigen specific stimulation, we generated a new K562-based artificial antigen presenting cell line (aAPC) expressing EGFRvIII lacking its intracellular domain (K562-EGFRvIII654, called KEAS). We have engineered another EGFRvIII expressing new aAPC line using a K562 cell line already stably expressing additional co-stimulatory molecules CD32, CD80, 41bbl (K562CD32-CD80-41bbl-EGFRvIII654, called KJEAS). We are currently testing KEAS or KJEAS feeder cell lines by co-culturing G1 or G3 T cells with neither CD3 or CD28 stimulation, nor IL-2 supplementation, and investigating IL-2 and IFN-gamma production of G1 or G3 T cells.

Cytokine stimulation and the choice of promoter are critical factors for the efficient transduction of primary mouse T-cells with HIV-1 based lentiviral vectors David Gilham 1; Michael Lie-A-Ling 2; Naomi Taylor 3; Robert Hawkins 1 1University

of Manchester, Medical Oncology, Manchester, United Kingdom; 2Paterson Institute for Cancer Research, Gene Therapy, Manchester, United Kingdom; 3Universite Montpellier, Institut de Genetique Moleculaire de Montpellier, CNRS, Montpellier, France Mouse T-cells are resistant to HIV-1 at multiple levels of the cycle of infection (1,2) In order to explore whether this precluded the use of HIV derived lentiviral gene therapy vectors for mouse T-cell transduction, splenic T cells were cultured with eGFP encoding 3rd generation lentiviral vectors (3) at an m.o.i of 10:1 and the T cells activated using antibody coated magnetic beads and the cytokines IL-2 and IL-7. After five days eGFP positive T cells were clearly identified. However, the promoter was critically important for expression with the rank order of potency of promoters as follows: human PGK human EF1SFFV CMV. Expression was maintained for at least one month after adoptive transfer of low numbers of gene-modified T cells into 6 Gy irradiated recipient mice. Importantly, a retroviral vector pseudotyped with the VSV-g envelope failed to efficiently transduce mouse T cells when similarly added at the start of the activation process. The lentiviral vectors used included a human PRE region though replacing this region with a woodchuck PRE did not abolish primary mouse T cell transduction. There was no apparent difference in the sensitivity of CD4 or CD8 T cells to lentiviral transduction. Furthermore, primary splenic T cells stimulated only with IL-2 or IL-7 were also transduced albeit with a lower relative level of transgene expression. Given the correct choice of promoter, primary mouse T cells are suitable targets to explore lentiviral gene therapy approaches. (1) J Virol. 2007 Jan;81(2):677-88. (2) J Virol. 2004 Nov;78(22):12537-47. (3) J Hepatol. 2002 Apr;36(4):459-65. Supported by the FP6 programme ‘ATTACK’. E-mail: [email protected] P 113 The “3rd generation” chimeric immune receptor engineered T cells: Can we improve adoptive immunotherapy strategies against glioblastoma? Ayguen Sahin ; Steven Maxfield ; Szofia Bullain ; Carlos Sanchez ; Elisabeth Baratta ; Bob S. Carter Massachusetts General Hospital-Harvard Medical School, Neurosurgery, Boston, MA, United States The CIR (chimeric immune receptor) technology is based on the redirection of immune effector cells toward a selected target using chimeric receptors that are linked directly to the signaling domain of the endogenous T cell receptor (TCR). Using this technology, we previously engineered a “1st generation” CIR targeting EGFRvIII mutant protein (MRI-CIR or G1). We have recently demonstrated the ability to retarget T cells to kill EGFRvIII expressing human glioblastoma cells in vitro and prolong the survival of mice bearing such tumors upon adoptive transfer in vivo. We conclude that MRI might be an effective anti-tumor strategy specific for glioblastoma. However, despite these encouraging results, our data in vivo also suggest that the lifespan of these CIR

E-mail: [email protected] P 114 Mouse models for gene therapy of HIV-infection Janine Kimpel ; Marianne Hartmann ; Felix Hermann ; Sebastian Newrzela ; Dorothee von Laer Georg-Speyer-Haus, Applied Virology and Gene Therapy, Frankfurt, Germany Background: Drug toxicity and viral resistance limit longterm efficacy of antiviral drug treatment for HIV infection. Thus, alternative therapies need to be explored. Previously, the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses a membrane-anchored HIV entry inhibitor (maC46) was tested in a Phase I clinical trial. Gene-modified autologous T cells were infused into 10 HIVinfected patients with advanced disease and multidrug resistant virus during antiretroviral combination therapy. Transfer of gene-modified cells was safe, led to sustained levels of gene marking and induced some improvement of immune functions. Here, the efficacy of maC46 was further investigated in a humanized mouse model. Methods: Human CD4 T cells isolated from healthy donors were transduced with gammaretroviral or lentiviral vectors encoding maC46 or a control peptide. After transduction cells were infected with a highly pathogenic primary HIV-isolate. The next day, HIV-infected, gene-modified T cells were transplanted into immunodeficient NOD/ SCID/gamma chain knockout mice. Results: Virus replication was quantified in the serum of the transplanted mice via RT-PCR. Expression of transgenes was monitored in human CD4 T cells in peripheral blood and spleen by FACS-staining. There was a substantive increase of maC46-expressing CD4 T cells in blood as well as in the spleen apparently due to the selective pressure of ongoing HIV infection .This increase of CD4 T was not seen in uninfected control animals nor with the control vector. Unlike the change in CD4 T cells, there was no significant difference in viral load between the maC46 and the control group, which is likely to be due to the limited observation period in this short term model. Conclusion: The clear accumulation of gene-protected cells in HIV-infected humanized mice indicates that these cells are protected from the effects of HIV infection in vivo.

ESGCT 2008 POSTER PRESENTATIONS

1141

Further studies in this model should allow us to analyze the conditions that determine efficacy of T cell based immuno/gene therapy for HIV-infection.

Experimental Hematology, Hannover, Germany; 3Johann Wolfgang Goethe University, Dep. of Exp. Paediatric Haematology and Oncology, Frankfurt, Germany

E-mail: [email protected]

Graft-versus-Host-Disease (GvHD) is the single largest factor of post allogeneic stem cell transplantation morbidity and mortality. Although the phenomenon can be largely abrogated by pre-infusion T cell depletion, such measures result in loss of engraftment capacity and reduced anti-viral and anti-leukaemic effects. The genetic modification of T cells with a suicide gene has been proposed as means of allowing T cells to be exploited for their beneficial effects, and safely eliminated in the event of causing significant GVHD. We developed a retroviral RSF91 suicide gene vector by systematically comparing modes of action of co-expression. Cells expressing the OuaSelect selection marker and thymidine kinase (TK) either as a fusion gene complex, from one transcript with 2A peptide linkers, or with an IRES were analyzed. Interestingly, only the fusion complex with the TK gene at the first position allowed for a highly correlated functional activity of the two genes. In primary T cells, the 48 hour selection resulted in a 99% pure population that was eliminated to 95% by exposure to 3g/ml of ganciclovir for 3 days. Thus, we were able to construct a vector that offers a novel attractive selection marker/suicide gene combination for T cell suicide gene therapy.

P 115 Evaluation of AZT for suicide gene therapy Anja Stoffel ; Anja Graven ; Remo Perozzo ; Leonardo Scapozza University of Geneva, EPGL, Quai E. Ansermet, Geneva, Switzerland Background: Herpes simplex virus Thymidine Kinase (HSV1-TK) together with Gancyclovir (GCV) is a well investigated as a failsafe for allogeneic haematopoietic stem cell transplantation (allo HCT). Patients who received donor T lymphocytes transduced previously with a retroviral vector coding for HSV1-TK, were successfully treated with GCV in case of Graft versus Host Disease (GvHD). Despite the demonstration of the efficacy of HSV1TK/GCV suicide strategy, this system is limited by the viral origin of the suicide gene, which can lead to immune mediated elimination of the engineered lymphocytes. The aim of our project, in order to improve the treatment, is the development of a new suicide gene/prodrug system with reduced immunogenicity. An engineered human enzyme, modified in its ability of activating prodrug would thus represent an interesting candidate for suicide gene therapy (SGT). Therefore we focused our study on the development of a fusion protein (fp) of the modified human cytosolic thymidine kinase 1 (hTK1) with the modified thymidylate kinase (TMK), with an enhanced ability to phosphorylate nucleoside analogue prodrugs and a reduced specifity for thymidine (dT). Methods and Results: hTK1 carrying different mutations were tested for their ability to phosphorylate AZT as well as dT. One promising candidate emerged from this screening. The substrate specificity of the hTK is shifted towards the prodrug. The modified hTK was fused with the modified TMK and thus 143B osteosarcoma cells lacking the hTK1 were transfected with the engineered enzyme. After treatment with AZT the cell viability was measured, revealing that the sensitivity against the prodrug for the transfected cells is around 100 fold higher then compared with cell carrying the hTK wild type. Jurkat cells were transduced with the fusion protein (lentiviral transduction). Cell viability was tested upon AZT treatment, revealing no significant difference between mock and fp transduced cells. Although the protein was expressed (WB), and high accumulation of AZTTP could be monitored. Conclusion: AZT toxicity is coupled more with NTP depletion then with the direct toxic effect of AZT-TP, thus the system might be more useful in HIV therapy then in SGT. E-mail: [email protected] P 116 Optimal ouaselect and thymidine kinase functionality in T cells is defined by the transgene cassette Alexandra Treschow 1; Axel Schambach 2; Ulrika Felldin 1; Evren Alici 1; Gösta Gahrton 1; Boris Fehse 3; Christopher Baum 2; M. Sirac Dilber 1 1Karolinska

Institutet, Department of Medicine, Stockholm, Sweden; 2Hannover Medical School, Department of

E-mail: [email protected] P 117 Sustained antigen presentation after lentiviral immunization Frederick Arce 1; Helen Rowe 1; Luciene Lopes 2; David Escors 2; Benjamin Chain 2; Mary K Collins 2 1Windeyer

Institute - University College of London, Department of Immunology and Molecular Pathology, London, United Kingdom; 2University College of London, Immunology and Molecular Pathology, 46 Cleveland Street, London, United Kingdom Lentiviral vectors have been used for antigen gene delivery into dendritic cells (DCs) to elicit immune responses. For example, vaccination with lentivectors expressing tumour antigens can generate specific cytotoxic and helper T cells that protect mice against tumour challenge. Previous studies have shown that DCs have a short life span and that the process of antigen presentation is self-limited. Since these vectors produce stable and high level expression of the transgenes, an important question is whether they could lead to a sustained antigen presentation. The presence of stably transduced DCs could either generate sustained immunity or tolerance to the expressed antigen. With this aim, transduced splenic DCs were tracked in vivo using GFP as a reporter gene. Different subsets of GFPpositive DCs were present even 30 days after mice had been injected intravenously with lentiviruses. Whether this sustained expression of the transgene in DCs is due to infection of long-lived mature DCs, immediate precursors or a case of cross-priming from the initially infected cells still remains to be answered. To determine if the transduced cells were still presenting antigen, mice vaccinated with lentivectors carrying the transgene for ovalbumin (OVA) were adoptively transferred with CFSE-labelled OT-1 cells. The latter are transgenic CD8 cells that carry the T cell receptor specific for the OVA epitope. One month after vaccination with the lentivector, there was still expansion of

1142 OT-1 cells in vivo, showing that the antigen was still being presented. Further experiments are being performed to determine if this sustained antigen presentation not only expands but also activates the specific T cells against the antigen both in vivo and in vitro. E-mail: [email protected] P 118 Lowering the risk of insertional mutagenesis for cell vaccine development: Non-integrating tricistronic lentiviral vector co-expressing HSV-TK, GM-CSF and IL-4 promotes effective self-differentiation of hematopoietic precursor cells into dendritic cells and subsequent scheduled elimination with GCV Mudita Pincha 1; Bala Sai Sundarasetty 1; Axel Schambach 2; Christopher Baum 2; Arnold Ganser 1; Renata Stripecke 1 1Hannover

Medical School, Hematology, Hannover, Germany; Medical School, Experimental Hematology, Hannover, Germany 2Hannover

ESGCT 2008 POSTER PRESENTATIONS P 119 Assessment of adenoviral vectors in gel formulations in vitro and in vivo: Aiming towards development of an HIV-1 vaccine Julian Harris 1; Takis Athanasopoulos 1; Veronique Bachy 2; Timos Papagatsias 3; Adel Benlahrech 3; Eszter Csomor 2; Andrea Meiser 3; Nick Roesen 4; Sea-Jin Oh 5; Len Seymour 4; Kerry Fisher 4; Rod Daniels 6; Romina Barbagallo 3; Steven Patterson 3; Linda Klavinskis 2; George Dickson 1 1Royal

Holloway, University of London, Biological Sciences, Egham, United Kingdom; 2King’s College London School of Medicine, Peter Gorer Department of Immunobiology, London, United Kingdom; 3Imperial College London, Department of Immunology, London, United Kingdom; 4Hybrid Therapeutics Ltd, Genetics, Oxford, United Kingdom; 5TheraJect, Inc, Development, California, United States; 6The National Institute for Medical Research (NIMR), VLP Development, London, United Kingdom

Background: Approaches to facilitate the generation of highly viable and potent Dendritic Cells (DCs) for large-scale vaccination clinical trials are warranted. We have previously demonstrated that mouse bone marrow cells transduced with a lentivirus vector (LV) for GM-CSF and IL-4 co-expression and directly injected s.c. into mice achieved self-differentiation into DCs in vivo, persisted for a long time ( 40 days) in the injection sites and resulted in high influx of DCs into lymph nodes. C57BL/6 mice vaccinated with LV/DCs co-expressing melanoma antigens showed potent protective and therapeutic anti-melanoma effects (Koya et al, Molec. Ther. 2007). Here, our goal was to lower the risk of lentiviral insertional mutagenesis for clinical development of LV/DCs. Methods: We constructed tricistronic lentiviral vectors with the ubiquitous CMV promoter or the antigenpresenting cell restricted MHCII promoter driving coexpression of HSV-TK, GM-CSF and IL-4. Integrating (IL) and non-integrating (NIL) lentiviral vectors were produced at high titers. Mouse bone marrow cells were transduced with the vectors and kinetics of DC differentiation was followed by expression of immunophenotypic markers. Cell killing was evaluated by viable cell counts and propidium iodide staining after addition of GCV (200 mM) to the cells. Results: Interspacing 2A elements allowed co-expression of the three gene products. Immunophenotypic analyses of CD11c, CD11b, MHCII, MHCI demonstrated that bone marrow cells transduced with the different type vectors self-differentiated into DCs, albeit with different efficiencies (CMVIL MHCII-IL CMV-NIL MHCII-NIL). Analyses of the CD11c/MHCII differentiation markers showed approximately 43-62% DC purity at day 7 post-transduction, increasing subsequently to 72-94% with time (days 14, 21). 70% of the DC populations expressed the CD83 maturation marker. GCV added to the culture at day 7 after transduction resulted into effective DC killing ( 90% at day 21 for the CMV-IL and CMV-NIL vectors). Analyses in vivo are ongoing. Conclusion:We demonstrated the feasibility of programming self-differentiation of DCs with safety enhanced lentiviral vectors, for lowering the risks of insertional mutagenesis.

Background:This study aims to optimise stability of serotypes 5 and 11 adenoviral vectors in various sugar formulations for the manufacture of an adenovirus vector vaccine against HIV-1. The vaccine will deliver modified HIV-1 genes and will take the form of a dry micro-needle array skin patch to target cutaneous Langerhans DCs to stimulate both systemic and mucosal immune responses against HIV-1. Methods: rAd vector stability and infectivity studies were performed by mixing either Ad5/Ad11-CMV-eGFP vectors with different sugar/gel formulations. The formulations included either: sucrose, trehalose, mannose, raffinose, stacchyose, inositol, maltodextrin, or lactose sugars, in SCMC or in combinations thereof. Sugar micro-needles containing the embedded vector were formulated into a patch comprising 25-30 needles up to 1500 m in length (micro-needles) by embedding the vector in the needle core maintaining the necessary penetration properties. Results/Conclusion: In order to develop the platform of the technology for micro-needle arrays/ patches for skin vaccination, 3 major areas of research have been investigated, namely: 1. Selection of an optimal skin vaccination regimen 2. Stability and infectivity of dessicated rAd vectors in vitro and in vivo 3. Formulation of rAd vectors into a powdered matrix Initial studies for delivery of the vector, as presented herein have analyzed and identified sugar mixtures (i.e 30% sucrose/8% SCMC) that give optimal preservation of infectivity whilst maintaining the strength and rigidity of the micro-needle array. 1-3 month stability data showed a progressive loss of infectivity for both rAd5 and rAd11 vectors in gel formulations over time, when compared to previous 1 day/1 week data, but still vectors retain substantial infectivity (i.e 55% eGFP  cells). In vivo stability/ infectivity studies are currently being assessed via transcutaneous injections of dessicated Ad-OVA-GFP vectors (4  10E9vg) in C57BL/6 mice (n  40) and foot-pad mediated migration of Ad5-CMV-eGFP to lymph nodes (n  24). Data produced by these studies will not only be important for the formulation of a micro-needle skin patch vaccine against HIV-1, but also for long term preservation and dissemination studies of viral vector formulations at ambient temperatures.

E-mail: [email protected]

E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS

1143 E-mail: [email protected]

P 120 Ubiquitination and segmentation of HIV genetic components: Aiming towards an HIV vaccine inducing broad(er) T cell immune responses 1;

2;

Takis Athanasopoulos Timos Papagatsias Julian Harris 1; Veronique Bachy 3; Adel Benlahrech 2; Eszter Csomor 4; Andrea Meiser 2; Nick Roesen 5; Sea-Jin Oh 6; Fusheng Li 7; Deborah Donnell 7; Steve Self 7; Sung-Yun Kwon 6; Kerry Fisher 5; Len Seymour 5; Rod Daniels 8; Linda Klavinskis 4; Romina Barbagallo 2; Steven Patterson 2; George Dickson 1 1Royal

Holloway, University of London, Biological Sciences, Surrey, TW20 0EX, Egham, United Kingdom; 2Imperial College London, Department of Immunology, London, United Kingdom; 3King’s College London School of Medicine, Peter Gorer Department of Immunobiology, London, United Kingdom; 4King’s College London School of Medicine, Peter Gorer Department of Immunobiology, London, United Kingdom; 5Hybrid Therapeutics Ltd, Genetics, Oxford, United Kingdom; 6TheraJect, Inc, Development, California, United States; 7Fred Hutchinson Cancer Research Center, Vaccine Immunology Statistical Center (VISC), Seattle, United States; 8The National Institute for Medical Research (NIMR), VLP Development, London, United Kingdom Background: Development, evaluation and optimisation of HIV-1 genetic vaccine components to generate a large pool of CD8 T cell memory cells recognising multiple CTL epitopes is currently one of the primary branch strategies in anti-HIV-1 vaccination protocols. This project, aims to develop viral/non-viral vector vaccines against HIV-1 by targeting skin dendritic cell (DC) populations. Methods: Vector delivery will be facilitated by sugar micro-needle patches which will be used to deliver to the skin a cocktail of adenoviral vectors carrying HIV-1 antigens. Initial studies have analyzed sugar mixtures that give optimal preservation of vector infectivity. Ubiquitinated full sized or segmented gag vectors were flanked at the C-terminus by an HA-tag signal to facilitate detection of HIV-1 genetic components as analysed by FACS and Western blotting. Results/Conclusion: We aimed to induce immunity to HIV-1 infection by developing a cohort of vectors in which HIV-1 vaccine components have been engineered and modified to stimulate broader CTL responses. In conjunction, two main approaches were envisaged, namely, genetic ubiquitination and segmentation of HIV-1 genes. Antigen ubiquitination results in its efficient proteasomal degradation whereas genetic fragmentation reduces peptide competition for available MHC molecules on the surface of the antigen presenting cell. Full-size HIV-1 i.e. gag genes from two HIV1 strains (CN54 and 96ZM651) were engineered, fused to mono- or tetra-ubiquitin (Ub) sequences and further tested on DC and non-DC cell lines by using plasmid, in vitro synthesised mRNA, or Adenoviral (5 or 11) vectors. In the segmentation approach a codon optimised gag CN54 gene has been fragmented into 10 separate segments and subcloned as N-linked monomeric or tetrameric Ub fusions. The segments were designed to contain similar numbers of epitopes (on average 4.6 epitopes each) aiming to overcome competition for antigenic presentation. I.m, plasmid electoporation (2x25 gEP) vaccine regimes (n  30) or adenoviral i.m./ transcutaneous injections (4  10E9vg) (n  40) of Ub/segmented gag/reporter genes in C57BL/6 mice aiming to corroborate observed data that ubiquitin (micro or full) gag fusions target the proteasome more effectively, are currently being assessed.

P 121 Optimisation of immunotherapeutic protocols using genetically modified cellular vaccines in a murine model for HPV16-associated tumours Milan Reinis ; Marie Indrova ; Jana Simova ; Jan Bubenik Institute of Molecular Genetics, AS CR, Tumour Immunology, Prague, Czech Republic Background: Genetically modified tumour cells represent an attractive immunotherapeutic tool for cancer treatment. For optimisation of the treatment, immunotherapy can be used in combination with tumour debulking, either by surgery or chemotherapy, as well as with treatments targeting immune suppression. Methods: Using a murine model for HPV16-associated tumours (TC-1 tumour cell line and MHC class I-deficient derivative TC-1/A9), we have investigated the therapeutic effects of IL-12-producing cellular vaccines for the treatment of minimal residual tumour disease after surgery or chemotherapy with CBM-4A ifosfamide derivative. This therapy was combined with depletion of T regulatory cells with antiCD25 monoclonal antibody, as well as with -galactosyl ceramide, a potent modulator of NKT cells mechanisms. Results: We have demonstrated the therapeutic effects of IL-12-producing cellular vaccines for the treatment of minimal residual tumour disease. Further analyses of the immune responses and changes in tumour infiltrating leukocytes have shown that treatment with IL-12 restored cytotoxicity and proliferative potential of CD45 tumour-infiltrating cells (TIL), increased tumour infiltration with CD8 and CD4 cells and decreased the Gr-1/CD11b cells in mice carrying syngeneic TC-1 and TC-1/A9 after chemotherapy with CBM-4A. Further, we have shown synergistic effects of immunotherapy of recurrent tumours with IL-12 producing tumour cells combined with depletion of T regulatory cells or with -galactosyl ceramide treatments. Conclusion: Our data demonstrate the therapeutic potential of the cell therapy with IL-12 producing tumour cell lines. The data suggest that the IL-12-producing cellular vaccine can restore the cytolytic potential and inhibit immunosuppressive TIL-dependent mechanisms in the individuals bearing HPV 16-associated tumours. Further, this treatment can be combined with targeting of Treg and NKT cells in order to optimize the immunotherapeutic protocols. Acknowledgement: This work was partly supported by the EU Clinigene Network of Excellence for the Advancement of Gene Transfer and Therapy and by the Grant Agency of the Czech Republic, Grant Nos. 301/06/774 and 301/07/1410. E-mail: [email protected] P 122 A novel, codon-optimised HSVtk(A168H) mutant for suicide gene therapy Ellen Preuß 1; Alexandra Treschow 2; Jürgen Otte 1; Sirac Dilber 2; Boris Fehse 3 1Frankfurter Stiftung für krebskranke Kinder, Experimental Paediatric Oncology and Haematology, Frankfurt am Main, Germany; 2Karolinska Institute, Department of Medicine, Division of Hematology, Huddinge, Stockholm, Sweden; 3University Hospital of the Goethe-University, Experimental Paediatric Oncology and Haematology, Frankfurt am Main, Germany

1144 The Herpex simplex virus thymidine kinase (HSVtk) gene together with its prodrug Ganciclovir (GCV) is the currently most widely used tool for both adoptive immunotherapy with suicide gene-modified donor lymphocytes after allogeneic stem cell transplantation and cancer gene therapy. Problems include unspecific toxicity of elevated GCV doses (e.g. immunosupression) and toxic side effects of high HSVtk levels in transduced cells (due to phosphorylation of cellular targets). Previously, the HSVtk(A168H) mutant was shown to have strongly reduced affinity towards its natural substrate thymidine. Here we developed a codon-optimised (co-)HSVtk(A168H). Performance of the latter was assessed in comparison to wild-type (wt) HSVtk and a novel, codonoptimised version of wtHSVtk. Firstly, all genes were fused to the established tCD34 selection marker to create sort-suicide genes. Those were in parallel introduced into various target cells via retroviral MP71 vectors. Transduced cells were magnetically sorted (MACS) to obtain pure population. For cell killing, different concentrations of GCV (1g/ml and 4g/ml) were applied for up to 1 week. We found strongly improved killing kinetics for the co-HSVtk A168H mutant as compared to both wt-HSVtk and co-wtHSV-tk in both myeloid (K562) and lymphoid (PM1) cell lines. This data was confirmed in primary human T cells. Notably, the differences became particularly apparent at lower GCV doses. Also, long-term expression data showed a clear advantage of coHSVtk A168H over the other TK genes in all cell types tested indicating reduced toxicity of suicide gene overexpression. Finally we assessed the potential of co-HSV-tk A168H in killing cancer cells. Different tumour cell lines were transduced with LeGO vectors expressing co-HSVtk A168H or one of the control genes in conjunction with sort-selection markers. Again, the highest killing rates after GCV-treatment were found for the co-HSVtk A168H gene at significantly lower GCV doses as compared to the controls. In summary, we provide evidence that the novel codon-optimised HSVtk A168H gene shows improved efficacy and lower toxicity as compared to wt-HSVtk co-wtHSVtk. This gene therefore represents a promising alternative for various types of suicide gene therapy. E-mail: [email protected] P 123 Ectopic expression of the extracellular domain of Mpl is sufficient to induce a hematopoietic population crisis Ute Modlich 1; Daniel C. Wicke 1; Johann Meyer 1; Guntram Buesche 2; Hans Kreipe 2; Zhixiong Li 1; Matthias Ballmaier 3; Christopher Baum 1 1Hannover

Medical School, Experimental Hematology, Hannover, Germany; 2Hannover Medical School, Institute of Pathology, Hannover, Germany; 3Hannover Medical School, Pediatric Hematology and Oncology, Hannover, Germany The thrombopoietin receptor Mpl is required for proper regeneration of hematopoietic stem cells and governs megakaryocytic differentiation. Patients with inherited MPL deficiency suffer from severe thrombocytopenia which progresses to aplastic anemia. As a first step towards a potential gene therapy for MPL deficiency, we retrovirally expressed the Mpl receptor in a murine bone marrow transplantation model. An initial series of vectors used a strong enhancer-promoter derived from spleen-focus forming virus (SFFV). Mice transplanted with hematopoietic cells modified by these constructs developed a profound yet transient elevation of multi-lineage hematopoiesis presenting as

ESGCT 2008 POSTER PRESENTATIONS chronic myeloproliferative disorder (CMPD). A minority of mice (3/27) developed erythroleukemia, which could be explained by insertional mutagenesis of Sfpi1, Fli1 and Klf3. Somewhat unexpectedly, in the majority of mice the CMPD converted into a progressive, potentially lethal pancytopenia. This population crisis affected all major blood lineages and also involved co-existing unmodified hematopoiesis thus causing a myelodysplastic syndrome (MDS)-like disorder. In the bone marrow, the MDS-like disorder presented with a loss of the primitive cell fraction (LSK cells). To address the mechanism of pancytopenia, we expressed a dominant negative form of Mpl (dnMpl) which lacks the intracellular signal transduction domain. Animals transplanted with dnMpl-modified cells failed to show the initial CMPD but developed the same pancytopenic, MDS-like end stage. A vector expressing Mpl under control of the PGK promoter or a fragment of the Mpl-promoter reduced or completely avoided the side effects observed with vectors using stronger promoters. The induction of a hematopoietic population homeostasis thus depends upon Mpl expression levels, indicating the need for strictly regulated transgene expression in gene therapy for MPL deficiency. As ectopic expression of the extracellular domain is sufficient to cause MDS, sequestration of Tpo may contribute to the pathogenesis of this disorder. This study demonstrates that ectopic expression of a hematopoietic growth factor receptor may disturb organ homeostasis through interference with extracellular mechanisms of cell communication. E-mail: [email protected] P 124 Marking of stem cells with promotor-deprived selfinactivating gammaretroviral vectors Kerstin Cornils 1; Claudia Lange 1; Axel Schambach 2; Martijn H. Brugman 2; Regine Nowak 3; Michael Lioznov 1; Christopher Baum 2; Boris Fehse 3 1University

Medical Center Hamburg-Eppendorf, Clinic for Stem Cell Transplantation, Hamburg, Germany; 2Hannover Medical School, Department of Experimental Haematology, Hannover, Germany; 3Hospital of the Johann Wolfgang GoetheUniversity, Experimental Pediatric Oncology and Hematology, Frankfurt am Main, Germany Based on their ability to stably integrate in their host cell’s genome, gammaretroviral vectors have successfully been used for gene marking as well as gene therapy, mainly in the hematopoietic system. However, recently retroviral vectors were shown to influence growth and/or survival characteristics of hematopoietic stem cells (HSC) by insertional mutagenesis. This type of genotoxicity may result in clonal dominance, but also malignant transformation of affected clones. We used our established serial bone marrow transplantation (BMT) model to investigate whether gammaretroviral self-inactivating vectors deprived of an internal promotor (pd-SIN vectors) allow stable genetic marking of serially reconstituting murine HSC without the side effects described above. Two independent long-term transplantation experiments were performed. All transplanted mice showed normal hematopoiesis at final analysis after approx. 1 year observation. LM-PCR was used to establish the integration pattern of pd-SIN vectors which apparently resembles that of gammaretroviral LTR vectors in non-selected HSC with a preference for active genomic regions. The overall integration pattern was strongly different from that seen with gammaretroviral LTR vectors in the respective control

ESGCT 2008 POSTER PRESENTATIONS experiments performed in parallel. Insertions of pd-SIN into proto-oncogenes, growth-promoting and signaling genes occurred significantly less frequent. We also measured expression levels of genes adjacent to pdSIN integration sites by quantitative RT-PCR. For most genes (28/32) located in the vicinity of pd-SIN insertions only minor transcriptional dysregulations (below a factor of 3) were found. Upregulation of genes did not exceed a factor of 6. This data again significantly differed from earlier observations with LTR vectors where 1000 fold transcriptional up-regulation was observed in some cases. In conclusion, our data supports the use of pd-SIN vectors for gene marking, and suggests that SIN vectors lacking strong enhancers will also decrease the risk of clonal imbalance in therapy studies. E-mail: [email protected] P 125 Perturbed T and B lymphoid development in models of Wiskott Aldrich Syndrome induced by RNAi Laurence Jeanson-Leh 1; Els Verhoeyen 2; Véronique Parietti 1; Caroline Costa 2; Roseline Yao 3; Laetitia Van Wittenberghe 1; François-Loïc Cosset 2; Anne Galy 1 INSERM U790, Evry, France; 2ENS Lyon, INSERM U758, Lyon, France; 3Genethon, U790, Evry, France

1Genethon,

Wiskott Aldrich syndrome (WAS) is a rare genetic disease associating immunodeficiency with thrombopenia and eczema. The lack of WAS protein (WASp) in hematopoietic cells leads to multiple anomalies in cellular activation and migration. In particular, a T and B cell lymphopenia is observed in WAS patients but its central or peripheral origin remains unclear. Since patients cells are scarce, we developed an RNAi approach to knock-down WASp in normal human progenitor cells in order to study human WASp-deficient progenitor cells. We have characterized an efficient shRNA-encoding lentiviral vector (LVshRNA W7) which reproducibly inhibits more than 80% of WASp with 1 or 2 copies of integrated vector per human CD34 cell. To study the hemato-lymphoid development of WASp-deficient human progenitor cells in vivo, we engrafted human CD34 cells into f  c / , Rag2 / Balb/c immunodeficient mice following transduction with the LV-shRNA-W7. Ten to fourteen weeks post-engraftment, high levels of human cell reconstitution could be documented in some mice in the bone marrow, spleen or thymus. This was observed when CD34 cells were transduced with the W7 shRNA or with the control shRNA. In both cases, multiple lineages of cells such as various T and B-cell subsets, NK cells, myeloid cells were seen in the marrow, thymus or spleen confirming that the lack of WASp did not block central hematopoiesis. However, the thymopoiesis of WASp-deficient progenitor cells was abnormal. Thymocytes expressing high levels of W7-shRNA had elevated levels of CD3 while the double positive CD4 CD8 CD1a thymocyte population was often reduced and CD4 CD25 natural regulatory T cells seemed to be more prevalent than in controls. In bone marrow, the expression of high levels of W7 shRNA was associated with decreased B lymphopoiesis in some of the mice. This effect was confirmed in vitro in OP9 stromal co-culture assays. Three experiments reproducibly demonstrated that WASp deficiency in CD34 cells caused a strong reduction in the differentiation and production of CD19 CD10 B cells in vitro. Further studies are ongoing to understand the mechanisms involved and to correct these defects with gene transfer vectors. E-mail: [email protected]

1145 P 126 New lenti-vectors co-displaying RD114 and SCF target gene transfer to HSCs in vivo Els Verhoeyen ; Cecilia Frecha ; Caroline Costa ; Christelle Granier ; Didier Nègre ; François-Loïc Cosset INSERM U758, EVIR, ENS de Lyon, Lyon, France Background: Previously, we developed new VSVG/TPO- and VSV-G/SCF-co-displaying vectors that outperform conventional VSV-G pseudotyped lentiviral vectors (LVs) for ex vivo gene delivery to hematopoietic stem cells (HSCs). However, since the fusion glycoprotein VSV-G, is complement sensitive and its receptor is present on all tissues it is unsuited for in vivo targeting. Methods and Results: Therefore, we exchanged VSV-G for another fusion partner, a mutant cat endogenous glycoprotein, RDTR. The resulting RDTR/SCF- and RDTR/SCF/TPO-lentivectors were far more efficient in transducing hCD34 cells (up to 40%) than RDTR-LVs in absence of retronectin ( 0.5%). LVs for in vivo gene therapy need to be able to distinguish between the target cells of interest and non-target cells. We mimicked as close as possible an in vivo setting for targeting gene transfer to CD34 cells by transducing total cord blood containing only 0,001% CD34 cells. Importantly, RD/SCF-pseudotyped LVs efficiently targeted transduction to CD34 cells with 95-fold selectivity for CD34 cells as compared to T-cells. G/SCF-vectors transduced also the non-target T-cell population resulting in only 1.8-fold selectivity for CD34 cells transduction. We assesed in vivo targeted gene transfer into hCD34 cells by intra-marrow injection of humanized Rag2 / ;gammac / Balbc mice with a low vector doses (1E5 IU/ml). One week later we detected a selective transduction of up to 3% of early human progenitors (CD34 cells) and of 3% of the myeloid progenitors (CD13) in the BM. In contrast, monocytes and pre- and pro-B-cells were transduced to a very low extent. Importantly, we verified transduction of other hematopoietic tissues after in vivo administration of the vector. We did not detect GFP human thymocytes, B-cells or CD3 T-cells in the blood stream and we detected only low level B-cell transduction in the spleen. Conclusion: Summarizing, local administration of low doses of RD/SCFHA LV into the BM of humanized mice resulted in a selective transduction of hCD34 cells in vivo. E-mail: [email protected] P 127 Magselectofection - combined magnetic cell separation and magnetofection: A novel non-viral and viral technology for ex-vivo gene therapy Yolanda Sanchez Antequera ; Arzu Cengizeroglu ; Edelburga Hammerschmid ; Olga Mykhaylyk ; Christian Plank Klinikum Rechts der Isar, Institute of Experimental Oncology, Munich, Germany Magnetic cell separation is the method of choice for isolating specific cell populations from tissue samples. Magnetofection is a highly efficient transfection method exploiting the influence of magnetic field acting on non-viral or viral gene delivery vectors associated with magnetic nanoparticles. We examined whether Miltenyi MACS® magnetic cell separation technology and magnetofection can be combined into one integrated procedure of cell isolation and transfec-

1146

ESGCT 2008 POSTER PRESENTATIONS

tion. Magselectofection procedure was developed and optimised for both non-viral and viral vectors using Jurkat cells as a model. Magnetofection vectors were formulated with novel magnetic nanoparticles in combination with the Dreamfect-gold transfection reagent for non-viral gene delivery and with a third generation SIN lentivirus for viral gene delivery. Procedure for vector loading on Miltenyi columns was developed enabling up to 100% retention for both non-viral and viral magnetic complexes. Cell separation specificity, vector association with and internalization into target cells, cell viability and the efficiency and specificity of reporter gene expression were analyzed. Non-viral vector association with target cells 30 min post-incubation of the column on the magnet was 90% and vector internalisation 48 h post-magselectofection was 50%. For both nonviral and viral vectors, purity of targeted (Jurkat) cells postmagselectofection of a mixture of Jurkat and K562 (1:1) was 96  0.4%. Reporter gene expression was up to 29.2  1.3% and 70  4.7%, for non-viral and viral magselectofection, respectively and specific for targeted cells with cell viability higher than 80%. PBMC and MSCs from umbilical cord were successfully transfected using non-viral magselectofection yielding up to 5-15% and 31.5%  1.8 transfected cells, respectively. Proof of principle for magselectofection procedure is provided. This technology enables cell separation and transfection in a one-step, saves time and may become a useful tool for nucleic acid therapy approaches involving exvivo genetically modified cells. E-mail: [email protected] P 128 Chemotherapy pressure: Its impact on integration site patterns of lentivirally transduced human hematopoietic stem cells 1;

2;

3;

Nadja Grund Patrick Maier Uwe Appelt Heike Allgayer 4; Frederick Wenz 2; W. Jens Zeller 1; Stefan Fruehauf 5; Stefanie Laufs 4 1German Cancer Research Center, Pharmacology of Cancer Treatment, Heidelberg, Germany; 2Mannheim Medical Center, University of Heidelberg, Department of Radiation Oncology, Mannheim, Germany; 3University of Heidelberg, Department of Transplantation Immunology, Heidelberg, Germany; 4German Cancer Research Center, Molecular Oncology of Solid Tumors, Heidelberg, Germany; 5Paracelsus Klinik, Center for Tumor Diagnostics and Therapy, Osnabrueck, Germany

Background: Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34). Methods: Human CD34 cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine-DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34 cells were analyzed and compared with an in silico set of 106 random integrations. Results: We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34 cells and the in sil-

ico set: both groups showed (i) chromosomal preferences, (ii) a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, (iii) a random integration distribution regarding transcription start sites (TSS), and (iiii) most importantly no significant differences concerning the number of integrations in or near cancer genes. Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. Conclusion: The general distribution of lentiviral integrations in either untreated or treated human CD34 cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies. E-mail: [email protected] P 129 SLUG as a novel radioprotector of normal tissue by gene transfer using a lentiviral bicistronic SIN vector with floxed EGFP as selection gene Patrick Maier 1; Carsten Herskind 1; Stefan Fruehauf 2; W. Jens Zeller 3; Frederik Wenz 1 1Heidelberg University, Department of Radiation Oncology, Mannheim Medical Center, Mannheim, Germany; 2Paracelsus Klinik, Center for Tumor Diagnostics and Therapy, Osnabrueck, Germany; 3DKFZ, G402, Heidelberg, Germany

Tumour radiotherapy with large field irradiation results in an increase of apoptotic cell death of the radiosensitive haematopoietic stem cells (CD34). Upon irradiation p53 induces expression of SLUG which protects the damaged cell from apoptosis by directly repressing p53-mediated transcription of PUMA. Therefore gene therapy with SLUG as a transgene might protect bone marrow cells during radiotherapy. For this reason we cloned SLUG in a lentiviral SINvector as the first gene of a bicistronic expression cassette followed by a floxed IRES linker and EGFP as selection gene. In a first experiment TK6 cells (human lymphoblastoid cell line) were transduced with our SLUG-IRES-EGFP vector and irradiated with 0-6 Gy. 48 h later the proportion of GFP-positive (SLUG-expressing) cells was determined by FACS. We found an up to 3.8-fold increase in EGFP-positive cells. A pool of nearly 100% transduced cells was established by FACS-sorting for EGFP-expression. After irradiation with 05 Gy MTT assays were used to detect radioresistance levels in comparison to untransduced cells. SLUG-expression delivered a significant survival advantage. EGFP-expression was not responsible for this radioprotective effect since SLUG-expressing cells were also radioresistant after removal of EGFP by CRE-recombinase. This radioprotection could also be demonstrated in an AlamarBlue-proliferation assay after irradiation with 2 or 4 Gy and over a time period of 6 days where SLUG-expressing cells (with or without EGFP) had a survival advantage compared to untransduced cells. In the next step we irradiated human CD34 cells two days after transduction with the SLUG-vector. Measurement of portions of EGFP-positive cells 2 and 8 days later corroborated the radioprotective potential of lentiviral SLUG-overexpression (2- and 3.5-fold increase in EGFP-positive cells). These results suggest SLUG as a promising gene for gene

ESGCT 2008 POSTER PRESENTATIONS therapeutic normal tissue protection which might contribute to widening the therapeutic range in radiotherapy. E-mail: [email protected] P 130 Rapamycin-treated dendritic cells as tolerogenic cellbased therapy in arthritis Julie Quentin 1; Louis-Marie Charbonnier 2; Christian Jorgensen 3; Pascale Louis-Plence 1 1Inserm,

Inserm u844, université Montpellier 1, Montpellier, France; 2Université libre de Bruxelles, Institute for Medical Immunology, Bruxelles, Belgium; 3Inserm, Inserm u844, UM1, CHU Montpellier, Montpellier, France Background: Dendritic cells (DC) are professional antigen-presenting cells with unique properties to steer the outcome of immune responses. We previously demonstrated that repetitive injection of immature DC (iDC) protects mice from developing collagen-induced arthritis (CIA). Rapamycin is a macrolide antibiotic with potent immunosuppressive properties introduced in recent years as therapy in organ transplantation. The immunosuppressive activity of rapamycin has been ascribed primarily to its inhibitory effects on T cells. More recently the effect of rapamycin on differenciation, antigen uptake, and the immunostimulatory capacity of human DC was examined. In the present study we investigated the effect of rapamycin on the phenotype as well as the immunogenicity of iDC in mixed-lymphocyte reaction, and evaluated the tolerogenic potential of such rapamycin-treated DC in the CIA mouse model of arthritis. Methods: Bone marrow-derived DC were generated in the presence of 10ng/ml of rapamycin during 6 days. Maturation of rapamycin-treated DC was induced or not by addition of LPS. DC cytokine secretion profiles were quantified by ELISA and their phenotype monitored by facs analysis. In in vivo experiments, repetive injections of 5  105 rapamycin-treated DC was performed in naïve mice and regulatory populations monitored in various lymphoïd organs. In the CIA mouse model, repetitive injections of iDC or rapamycin-treated DC were performed at days 21, 23 and 25 after immunization. Results: The phenotype of rapamycin-treated DC was slightly different from conventional iDC, with a decreased expression of CD40 and co-stimulatory molecules such as CD86. The decrease of the immunogenicity of rapamycin-treated DC was confirmed in MLR. In vivo, we observed an increase of the IL10-secreting CD4CD49b T lymphocytes in the liver and spleen of the mice injected with the rapamycin-treated DC. In experimental arthritis, repetive injections of rapamycin-treated DC decreased the severity of established arthritis more importantly than conventional iDC. Conclusion: Our results suggest that the tolerogenic potential of iDC is increased when generated in presence of rapamycin. Such a cell-based immunomodulatory strategy might be assessed in RA. E-mail: [email protected] P 131 Allogeneic stromal cell implantation in brain tissue of immune-competent mice leads to robust microglial activation and subsequent graft elimination Bart R. Tambuyzer ; Irene Bergwerf ; Nathalie De Vocht ; Kristien Reekmans ; Jasmijn Daans ; Dirk K. Ysebaert ;

1147 Shyama Chatterjee ; Eric Van Marck ; Zwi N. Berneman ; Peter Ponsaerts University of Antwerp, Medicine, Antwerp, Belgium Aims of the Study: To investigate the immunological aspects of allogeneic bone marrow derived stromal cell (BMSC) transplantation in the central nervous system (CNS) of immune-competent mice. Methods: (I) In vitro: co-cultures of microglia (FVB) with autologous (FVB) or allogeneic (C57BL/6) BM-SC were screened for TNF- and NO production. (II) In vivo: BM-SC were injected intra-cranially followed by IFN- ELISPOT and histological analysis. Results: (I) In vitro: Both cell types used in this study were thoroughly characterised: (i) microglia displayed expression of CD11b, CD45low, F4/80, CXCR3 and CXCR4, and (ii) BMSC showed uniform expression of stromal cell markers Sca1, MHC I and CD106, but not MHC II and CD45. Microglia were stimulated to produce high amounts of TNF- and NO when stimulated with IFN-/LPS (100U, 500U/ml and 10 ng, 1 g/ml) while BM-SC only produced NO. Upon co-cultivation microglia nor BM-SC produced significant amounts of TNF- or NO. Moreover, if these co-cultures were further stimulated with the IFN-/LPS cocktail, no reduction or increase of TNF- production was detected, while NO secretion was only slightly increased. (II) In vivo: After implantation of allogeneic C57BL/6 BM-SC in the brain of immune competent FVB mice, we observed vigorous microglial activation up till 4 weeks post-implantation, demonstrated by CD11b staining. Simultaneously, the BM-SC marker Sca-1 diminished over time indicating graft elimination. Using CD3 staining, we did not detect significant T-cell infiltration. This finding was further supported by the absence of systemic immune responses against intra-cranial cell implants. In contrast, immunological rejection of intra-muscular injected allogeneic BM-SC was detectable by IFN- ELISPOT. Conclusions: (I) Microglia are not activated or inhibited by allogeneic BM-SC in vitro. (II) Rejection of allogeneic BMSC implanted in the CNS of immune-competent mice is mainly mediated by activated microglia without overt T-cell involvement. E-mail: [email protected] P 132 Strategies to overcome humoral immunity to AAV vectors in humans Margriet Vervoordeldonk 1; Federico Mingozzi 2; Shyrie Edmonson 2; Rogier Thurlings 3; Katherine High 2; Paul Peter Tak 3 BV, Arthrogen, Amsterdam, Netherlands; 2The Children’s Hospital of Philadelphia, Center of Cellular and Molecular Therapeutics, Philadelphia, United States; 3AMC/University of Amsterdam, Div Of clinical immunology and Rheumatology, Amsterdam, Netherlands 1Arthrogen

Background: Humoral immunity against AAV vector represents an important barrier to gene transfer, resulting in clearance of the vector before it enters the target cell. Antibodies directed to the AAV capsid are highly prevalent in humans, a natural host for this virus, and cross reactivity of antibodies among serotypes varies depending on the degree of homology of capsid proteins sequence. Both preclinical and clinical studies showed that antibodies against the AAV capsid block transduction at very low titer when the vector is introduced into the bloodstream, while gene transfer in

1148 other body districts, like the eye and the brain, seems to be less susceptible to neutralization. Method: In light of the development of intra-articular gene therapy for rheumatoid arthritis (RA), we measured neutralizing antibody titers against AAV serotype 2, 5, 6, 8 in serum and matched synovial fluid from RA patients. Results: Anti-AAV-2 antibodies were the most prevalent, with 9/11 subjects with a titer 1:10, compared to 3/11 for AAV-5, 7/11 for AAV-6, and 6/11 for AAV-8. Interestingly, antibody titers in synovial fluid were lower than in the matched plasma samples (average of 1714 vs. 2337 ng/ml IgG, respectively, p  0.05) indicating a difference in distribution of neutralizing antibodies to AAV depending on the body compartment. We next evaluated the impact on antiAAV antibodies of a B cell-targeted therapy in RA patients who received for the first time a single dose of anti-CD20 antibody as part of their disease management. Anti-AAV antibodies were measured at baseline, week 4, 16, and 24 postadministration; in all the samples analyzed, a drop of neutralizing titer was observed, in some subjects decreasing from as high as 1:31.6-1:100 to less than 1:3.16 by week 16 and returning to baseline levels by week 24. Conclusion: We have shown that serotypes other than AAV-2 may help escaping antibody neutralization, with AAV serotype 5 showing the lowest level of neutralization. Anti-CD20 antibody therapy results in a drop in anti-AAV antibody titer, potentially representing an additional modality to allow for vector administration in people with preexisting humoral immunity to AAV, or permitting vector readministration. E-mail: [email protected] P 133 Gene therapy promoting inflammation-triggered IL-10 production ameliorates collagen induced arthritis Louise Henningsson 1; Tove Eneljung 1; Ulf Lidberg 2; Fons van de Loo 3; Wim van den Berg 3; Andrej Tarkowski 1; Inger Gjertsson 1 1University

of Gothenburg, Dep.of Rheumatology and Inflammation research, Gothenburg, Sweden; 2Sahlgrenska Univeristy Hospital, Sahlgrenska Center for Cardiovascular and, Metabolic Research, Wallenberg Laboratory, Gothenburg, Sweden; 3University Medical Centre, Rheumatology Research and Advanced Therapeutic, Nijmegen, Netherlands Background: Rheumatoid arthritis (RA) is a chronic and destructive autoimmune disease which affects 1% of the western population and is characterised by flares. Current treatments are focused on constant suppression of the immune response irrespective of the inflammatory status. Interleukin (IL) -10 is a pleiotropic cytokine with anti-inflammatory properties. To enhance the endogenous levels of IL-10 during inflammation, an additional IL-10 gene, driven by an inflammation-inducible promoter consisting of the promoter region of IL-6 and the enhancer region of IL-1, has been inserted into the mouse genome by lentiviral vectors. This system would allow an arthritic host to increase the antiinflammatory regulation and inhibit episodes of flares whilst protecting form joint destruction. Methods: Collagen type II (CII) induced arthritis (CIA) is a mouse model of RA. Bone marrow derived hematopoetic stem cells were transduced with lentviruses containing IL10 or a control gene driven by the IL-6/IL-1 promoter, and injected into lethally irradiated DBA/1 mice. The mice were subsequently immunized with CII to induce arthritis which

ESGCT 2008 POSTER PRESENTATIONS was evaluated throughout the experiment, and tissues were examined post mortem. The mice were bled at different time points, and joints were taken for histopathological examination. Results: The mice treated with the IL-10 construct showed a delayed onset of disease and a less severe arthritis compared to controls. The frequency of arthritis in the control group increased to 70% while only 30% of the mice treated with IL-10 expressing virus were affected. No differences could be seen in IL-10 levels in blood, supernatants from lymph nodes, nor was there any differences in proliferation of lymph node or spleen cells between the groups. Conclusion: The use of viral vector expressing IL-10 delays the onset and ameliorates the severity and frequency of CIA. Further experiments are ongoing to elucidate the mechanism of protection. E-mail: [email protected] P 134 Clinical significance of TRAIL and its receptor expression profiles on CD4 CD25 Foxp3 Tregs and CD8 CD25 T cells in patients with rheumatoid arthritis Atil Bisgin 1; Veli Yazisiz 2; Mehtap Ulker 2; Ender Terzioglu 2; Salih Sanlioglu 1 1Akdeniz

University Faculty of Medicine, Depts. Of Medical Biology-Genetics and, Human Gene Therapy Unit, Antalya, Turkey; 2Akdeniz University Faculty Of Medicine, Department of Rheumatology—Allergy and, Immunology, Antalya, Turkey

Background: Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disorder that affects up to 1% of the population. The exact origin and pathogenesis of the disease are still unknown. The immune system plays a major role in both development and perpetuation of RA. Within the immune system of patients with rheumatoid arthritis, T cells have a central pathological function in both activating macrophages and B cells. Recent studies suggest that the immune abnormalities are not limited to the activation and clonal expansion of synovium-infiltrating lymphocytes, but involve the majority of circulating T cells. Increased apoptosis may induce autoimmune conditions like RA. Apoptosis is induced by binding of death receptor ligands, members of the tumour necrosis factor (TNF) superfamily, to their cognate receptors. TNF related apoptosis inducing ligand (TRAIL), ligand of the TNF superfamily, induces apoptosis in sensitive cells and essential for negative selection of T cells in the thymus and in the control of lymphocyte cell cycle. Methods: The frequency of CD4 CD25 Foxp3 regulatory T cells (Tregs) and CD8 CD25 T cells were determined by flow cytometry in 20 patients with RA and 20 healthy controls. Fluorescence-activated cell sorting (FACS) was used to sort CD4 CD25 Foxp3 T cells and CD8 CD25 T cells. Then the expressions of TRAIL ligand and its receptors on these peripheral blood lymphocytes of patients with RA and controls were analysed by flow cytometry. Results: In our previously reported study, synoviocytes of RA patients expressing various TRAIL receptors on the surface and these surface TRAIL receptor profiles mainly influence whether RA synoviocytes are sensitive or resistant to cell death by adenovirus mediated delivery of hTRAIL. Moreover, in this study we examined TRAIL and its receptor expression profiles of lymphocytes with CD4/CD25/Foxp3 and CD8/CD25 markers and

ESGCT 2008 POSTER PRESENTATIONS the frequencies of CD4 CD25 Foxp3 Tregs and CD8 CD25 T cells. It is observed that TRAIL and T cells have essential pathogenic roles in the disease activity of RA. E-mail: [email protected] P 135 Soluble TRAIL concentrations in patients with rheumatoid arthritis Atil Bisgin 1; Veli Yazisiz 2; Ender Terzioglu 2; Salih Sanlioglu 1 1Akdeniz

University Faculty of Medicine, Depts. of Medical Biology-Genetics and, Human Gene Therapy Unit, Antalya, Turkey; 2Akdeniz University Faculty of Medicine, Department of Rheumatology–Allergy and, Immunology, Antalya, Turkey Background: TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein that belongs to the TNF superfamily. Members of the TNF superfamily induce apoptosis. And increased apoptosis may induce autoimmune conditions. TRAIL activates rapid apoptosis in many transformed cells but not in normal tissues. Like TNF and FasL, TRAIL also exists physiologically in a biologically active soluble homotrimeric form. It has been previously reported that TRAIL could be involved in the pathophysiology of autoimmune diseases. In patients with systemic lupus erythematosus, serum soluble TRAIL (sTRAIL) concentrations are increased. The goal of this study is to investigate if the secreted sTRAIL might be important in the induction of apoptosis or the destruction of the joints in Rheumatoid Arthritis. Methods: Serum sTRAIL concentrations were measured by a solid phase sandwich enzyme linked immunoabsorbent assay. Levels in RA patients (n  20) were compared with those in patients with Sjogren’s Syndrome (n  20) and healty volunteers (n  20). The serum sTRAIL concentration in the whole groups were compared to the disease activity. Results: The exact origin and pathogenesis of RA are stil unknown, and numerous disease-modifying drugs and biologics have been tested. There is a significant need for increased efficacy and safety of these agents. And the Disease Activity Scale (DAS) 28 is not always a reliable indicator of treatment effect in RA. We examined the relation between sTRAIL concentrations and DAS score and drug treatment. The study showed various sTRAIL concentrations in the serum of RA patients compared with healty volunteers and patients with Sjogren’s Syndrome. Increased concentrations of sTRAIL are correlated to the treatment modalities which are known to induce apoptosis. E-mail: [email protected] P 136 Intraarticular gene transfer with the soluble type I TNFalpha receptor in collagen induced arthritis Anne Denys 1; Delphine Lemeiter 2; Natacha Bessis 1; Marie-Christophe Boissier 1

1INSERM

ERI-18, EA42-22, University of Paris 13, Bobigny, France; 2INSERM ERI-18, AP-HP Avicenne Hospital, Bobigny, France Local articular (i.a.) non viral gene therapy using electrotranfer (ET) is of interest in organ-targeted diseases such as rheumatoid arthritis (RA). It could permit to use lower quantities of plasmid vector, avoid adverse effects such as sys-

1149 temic toxicity, and allow to get higher levels of transgenic protein within the joint. Our objective was to evaluate in arthritic mice the therapeutic potential of a plasmid encoding the fusion protein formed with the soluble type I TNF receptor (sTNFRI) and the heavy chain of IgG1 (sTNFRI/IgG1). To induce arthritis, DBA/1 mice were injected with native collagen type II (CII) emulsified in complete Freund’s adjuvant. Then, the plasmid pVax1 mTNFR-Is/mIgG1 was injected into the right and left knee at time of clinical symptoms onset. After plasmid injection, an electric field was applied with electrodes placed apart on either side of the joint. After i.a. ET of various plasmid doses, a dose dependent expression was observed locally in knee-conditioned media and maximal secretion was observed after injection of 20 g chimeric plasmid. Local treatment of CIA with pVax mTNFR/mIgG1 electrotransfer induced significant decrease in the percentage of arthritic knees as compared to NaCl (37,5% versus 83.3%). An effect on non-knee joints was also observed 35 days (p  0.017) after first immunization with CII as compared to NaCl (10 days after ET). Plasmid alone has also a transitory effect on non-knee joint. We didn’t observe any significative difference of IL-17, VEGF, IL-4 and IL-6 expression in the synovial tissue. Intra articular gene therapy opens perspectives for non viral gene therapy applications to RA. Local effect of TNF soluble receptor is significant since it diminished joint inflammation and destruction. An experiment is in progress to evaluate cytokine expression after electrotransfer during whole arthritis course. These results demonstrate the feasibility and the effectiveness of local non viral gene therapy in arthritis. E-mail: [email protected]

P 137 Immunotoxicology and tissue response profiles of electro gene transfer (EGT) in skeletal muscle by microarray analysis Christopher Mann 1; Xavier Anguela 1; Joel Montane 1; Marta Moya 1; David Callejas 1; Luis Mir 2; Fatima Bosch

1

1Universitat

Autònoma de Barcelona (UAB), Centre de Biotecnologia Animal i de Teràpia Gènica, Ciber de Diabetes y Enfermedades Metabólicas, Bellaterra (Barcelona), Spain; 2Institut Gustave-Roussy, Univ Paris-Sud, Villejuif, France Electro gene transfer (EGT) is a common method for delivery of DNA into skeletal muscle to produce local effects, secretion of molecules or vaccination. Despite wide use of EGT, little is known about safety aspects associated with the electric field or the gene expression changes associated with immunological responses. Our goal was to perform a safety assessment of EGT using microarrays to analyse gene expression changes, in conjunction with histological analysis. The microarray data was used to create a set of marker genes that will be beneficial for comparing the safety of other EGT protocols and potentially other gene therapy approaches in muscle. We compared gene expression profiles in mouse muscle from non-electrotransferred controls, saline electrotransferred muscle and muscle treated with non-expressing plasmid in order to delineate the contribution of each process. We first demonstrate that the number and magnitude of gene expression changes in saline-EGT muscle were modest,

1150 in accordance with the limited infiltration and structural changes observed histologically. The majority of up-regulated genes were attributed to a muscle regeneration, largely in association with needle track damage. In contrast, EGT of a non-expressing plasmid produced large increases in the number and degree of gene expression in accordance with greater evidence of muscle injury and remodelling with substantial infiltration observed histologically. Importantly, many genes involved in the innate immune system recognition of nucleic acids were up-regulated suggesting that the innate immune system is responsible for triggering inflammation and that several new, but poorly characterised pathways may be involved. Overall, our results indicate that the EGT procedure itself is safe, although the presence of plasmid DNA is capable of exaggerating regenerative and innate immune responses which will provide insights into vaccine action and potential safety concerns for human trials using EGT. E-mail: [email protected] P 138 Hydrocatheter-interventionist gene transfer to specific organ in large animals Maria Jose Herrero 1; Salvador Lledo 2; Luis Sabater 2; Vicente Bodi 3; Luis Mainar 3; Maria Sanchez 1; Salvador F. Alino 1 1Universidad

de Valencia, Farmacologia, Valencia, Spain; Clinico Universitario Valencia, Cirugia, Fac. Medicina, Valencia, Spain; 3Hospital Clinico Universitario Valencia, Cardiologia, Fac. Medicina, Valencia, Spain 2Hospital

Background: . Hydrodynamic DNA injection in mice mediates highly efficient liver gene transfer by a mechanism that involves sinusoidal blood flow inversion and massive hepatocyte endocytic process. Our aim is to translate the systemic large volume i.v. injection (hydrodynamic) performed in small animals into a retrovenous injection of selected organ (hydrocatheterism) in large animals by interventionist catheterism, evaluating the efficacy of the procedure. Methods: Pigs were anaesthetized and catheterized through the jugular veins to reach the hepatic vein or coronary sinus, with two catheters, one with large flow capacity and the other with a balloon to hermetically close the vein. The correct positioning of both catheters was confirmed by fluoroscopy. Then, a plasmid saline solution (20g/ml) containing the human -1-antitrypsine (hAAT, for liver delivery) or green fluorescent protein gene (EGFP, for cardiac delivery) was injected at 10-20ml/s rate. After 3-5 minutes, the catheters were removed and the animals awaken, to be kept in the stall. Blood samples were taken and plasma kept at 20°C for quantitative ELISA. After sacrificed, samples of liver and cardiac tissue were collected for morphological and molecular studies. Results: Liver transfection experiments indicate that dosedependent hAAT protein in porcine plasma can be observed but the maximal effect is limited (aprox.500 ng/ml). The cardiac immunohistochemistry and quantitative RT-PCR results, after retrovenous injection of a plasmid containing EGFP gene in the coronary sinus, show that a wide gene expression can be reached in the muscular cardiac tissue, both in the auricular as well as in the ventricular territories. Conclusion: The hydrocatheter procedure shows that it is possible to transfer naked DNA to liver and cardiac tissue by retrovenous injection. However, the conditions of catheter implantation and perfusion flow must be adjusted in each

ESGCT 2008 POSTER PRESENTATIONS organ in order to optimize gene transfer efficacy. Project financed by SAF-2007-6449 and GV AP-071/08. E-mail: [email protected] P 139 Development of a large scale, non-viral platform for generation of redirected T-cells for adoptive transfer Hilde Almaasbak 1; Anne Marie Rasmussen 1; Gunnar Kvalheim 2; Johanna Olweus 1; Martin Pulé 3; Claudia Rossig 4; Gustav Gaudernack 1; Edith Rian 1 1Radiumhospitalet-Rikshospitalet,

Section of Immunotherapy, Institute of Cancer Research, Oslo, Norway; 2RadiumhospitaletRikshospitalet, Department of Cellular Therapy, Oslo, Norway; 3University College London, UCL Cancer Institute, London, United Kingdom; 4University Children’s Hospital Muenster, Department of Pediatric Hematology and Oncology, Muenster, Germany Background: Adoptive cell therapy using redirected Tcells represents a promising tool for cancer treatment. Integrating viral vectors are the major tools for genetic modification. However, electroporation of mRNA is considered a safe alternative when transient genetic modification is desired. Moreover, this method is less expensive and may provide a faster track to clinical studies as the regulatory regime is less demanding. The approach also allows for rapid screening of novel receptors prior to larger studies involving stably gene-modified cells. A technology platform for largescale clinical grade production of genetically modified T-cells by mRNA electroporation is warranted. Methods: T-cells were expanded for 10 days using Dynabeads ClinExVivo CD3/CD28 in the Wave Bioreactor. mRNA encoding a chimeric antigen receptor (CAR) directed against CD19 was electroporated into T-cells using the BTXECM830 Square Wave Electroporator. Receptor expression and potency of the electroporated T-cells were assessed by flow-cytometry (expression, degranulation and cytokine production) and Cr-release. Results: T-cells were expanded up to 700 fold and the majority of CD8 cells showed an early effector phenotype. More than 200 mill cells were transfected in one single cuvette and electroporation yielded high and uniform expression of CARs on nearly the entire cell population. The amount of mRNA for electroporation greatly influenced the expression level as well as the killing potential. Expression and lysis of CD19 tumor cells sustained for at least one week in vitro. Conclusion:We have combined a large-scale clinical grade Tcell expansion platform with mRNA electroporation technology to generate T-cells for the transient expression of chimeric antigen receptors targeting leukemia and lymphoma antigens. The platform is currently being set up for clinical studies. E-mail: [email protected] P 140 Influence of the chemical composition of amphiphilic triblock copolymers on muscle gene transfer Debborah Alimi 1; Blandine Brissault 2; Christian Leborgne 1; Christine Guis 2; Daniel Scherman 1; Antoine Kichler 1 FRE 3087 CNRS, Evry, France; 2Laboratoire Materiaux Polymeres Aux Interfaces, UMR CNRS 7581, Evry, France

1Genethon,

ESGCT 2008 POSTER PRESENTATIONS Amphiphilic triblock copolymers such as the polyethylene glycol-polypropylene glycol-polyethylene glycol L64 (PEG13-PPG30-PEG13) have been recently shown to promote muscle gene transfer. In the present study, we investigated the relation between structure of the copolymers and their capacity to enhance muscle transfection. To this end, we evaluated the in vivo activity of 6 copolymers that differ either by their molecular weight or by their hydrophilic/hydrophobic balance. Our results indicate that there is not a unique compound able to improve muscle gene transfer but there is a large flexibility in terms of molecular weight and EO/PO ratio. Further, we investigated the effect of a chemical change of the PEG moiety on the transfection activity. Therefore, we synthesized and characterized new amphiphilic copolymers in which the PEG end blocks were replaced by more hydrophilic poly(2-methyl-2-oxazoline) (PMeOXZ) chains of various lengths. The PMeOXZ-PPGPMeOXZ compounds were assayed for in vivo muscle gene transfer and the results indicate that they increased by 20fold the transfection efficiency as compared to naked DNA. Thus, the capacity to enhance DNA transfection in skeletal muscles is not restricted to PEG-PPG-PEG arrangements. Finally, we studied the membrane permeabilizing activity of these compounds by performing different membrane leakage assays in vitro and in vivo. E-mail: [email protected] P 141 Biodegradable PAMAM ester for enhanced transfection efficiency with low cytotoxicity Kihoon Nam ; Jong-Sang Park Seoul National University, School of Chemistry & Molecular Engineering, 599 Gwanak-ro, Gwanak-gu, Seoul, Republic of Korea In the previous study, we found that arginine-modified PAMAM dendrimers with amide bonds showed remarkable transfection efficiency and low toxicity compared with branched 25 kD PEI. However, the peptide bonds of the polymer may not be degraded very fast and be accumulated among tissues or cells for a time being retaining the polymer-mediated toxicity. To solve this problem, we designed and synthesized self-destroying polycationic polymers (ePAM-R, e-PAM-K, e-PAM-ahx, e-PAM-ahp) that contain amino-acids at the peripheral ends of PAMAM-OH dendrimer through ester bond linkages. The degradation profiles of the free polymers in D2O at 37 iAEC were determined by NMR spectroscopy. And then, the buffering capacity of the PAMAM polymers was examined by acid-base titration by adding 0.01 M HCl to polymer solutions. The PAMAM esters were readily degradable under physiological conditions (pH 7.4, 37 iAEC). However, polyplexes were very stable and were hardly degraded in the endosomal pH range. Moreover, these amino-acid-modified polymers showed excellent buffering capacities between pH 5.1 and 7.4, facilitating endosomal escape of polyplexes. The transfection efficiency of the newly synthesized biodegradable PAMAM esters was evaluated using the HepG2 cell line, and primary SMCs and HUVECs. While the lysine-grafted PAMAM ester and the aminohexanoic-acid-grafted PAMAM ester did not display significant improvement in transfection efficiency, the arginine-conjugated PAMAM ester-mediated transfection of a luciferase gene showed better transfection efficiency than the branched 25 kD PEI and PAM-R (peptide

1151 bond), and lower cytotoxicity, especially with primary cells such as HUVECs and SMCs. Furthermore, after DNA release, free e-PAM-R degraded completely into nontoxic PAMAM-OH and arginines by hydrolysis, which resulted in lower cytotoxicity in contrast to the poorly degradable arginine-modified PAMAM with amide bonds. These findings demonstrated that the arginine-grafted biodegradable PAMAM dendrimer, e-PAM-R, is a potential candidate as a safe and efficient gene delivery carrier for gene therapy. E-mail: [email protected] P 142 Internalization dynamics and localization of magnetic transfection complexes Anna Sauer 1; Karla de Bruin 1; Christian Plank 2; Christoph Braeuchle 1 1Ludwig-Maximilians-University

Munich, Department of Chemistry and Biochemistry, Munich, Germany; 2Institute for Experimental Oncology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany Magnetofection is a successful technique for gene delivery, where lipoplexes and magnetic particles with an iron oxide core are accumulated at target sites by means of a magnetic field. Although enhanced transfection efficiencies both in vivo and in vitro have been reported, few mechanistic data concerning cellular uptake and intracellular transport of the magnetic complexes are available. In this study a detailed description of the internalization and intracellular trafficking of magnetic lipoplexes was obtained by using highly sensitive fluorescence widefield microscopy. The movement of the complexes was observed on a single cell level starting with the attachment to the cell membrane, followed by the individual steps of the uptake process, and intracellular trafficking. The acquired image sequences were analyzed by single particle tracking obtaining trajectories representing the movement of the particles. In accordance with measurements on polyplexes [1] three modes of motion were observed: complexes attached to the membrane showed diffusion with drift, the second mode of motion was confined and anomalous diffusion and the third motion was classified as directed motion, most probably representing movement along microtubules. Apart from mechanistic information on the internalization process, the percentage of internalized complexes was determined at different time points. After 20 min 16% of the complexes are internalized, whereas after 80 min the percentage increases to 56% internalized complexes. Internalization via the endocytic pathway was determined for 96% of intracellular complexes via colocalization experiments with endosomal markers. After 1.5-3 h the formation of a perinuclear ring structure was observed, with subsequent accumulation of the complexes on one side of the nucleus. These data for the first time give a real-time view on the internalization and intracellular fate of magnetic lipoplexes. The obtained data suggest a similar transfection pathway for magnetic lipoplexes and polyplexes. Reference [1] De Bruin K, Ruthardt N, von Gersdorff K, Bausinger R, Wagner E, Ogris M, Braeuchle C, Cellular dynamics of EGF receptor-targeted synthetic viruses, Mol. Ther. 2007, 15, 1297-1305. E-mail: [email protected]

1152

ESGCT 2008 POSTER PRESENTATIONS

P 143

sistance sequences within vectors may result in detectable traces of these e.g. within deriving virus preparations. Especially for safety reasons it is necessary to avoid such sequence elements. Therefore, the European authority for the evaluation of medical products (EMEA) proposes in the guidelines for medical gene transfer products to avoid selection markers like resistances against antibiotics (CPMP/BWP/3088/99). The AB cassette (and other systems to select for the presence of plasmids within the host cell) is used in cultivation to serve as a selection marker. Deleting such structural element results in partial loss of plasmid carrying cells being overgrown by “empty” cells due to their higher specific growth rate. We currently develop a large-scale production process for non-viral DNA vectors without genes for the resistance against antibiotics and other selection markers: Minicircle-DNA. Methods: This vector system is based on a parental plasmid (PP) carrying a selective marker, the origin of replication (ori) for replication within the E. coli host cells, two recombination sites for cis-recombination and the “gene-of-interest” with all necessary elements for regulation in the target cells. The enzyme catalyzed recombination of a PP results in two circular supercoiled molecules: a miniplasmid (MP) containing the selection marker, the ori and all the unwanted and unessential bacterial sequences and the Minicircle (MC) with nothing but the gene-of-interest and a tiny residual sequence stretch resulting from the recombination. A sequence specific affinity chromatography is used to specifically separate the mini-plasmid from the MC as the basis for a large-scale purification process to manufacture this molecule for pre-clinical and clinical applications. Results: Within this study reporter genes for different types of analyses within various tissues, cells, animals and for testing the mode of administration were used. THe obtained results shown here demonstrate a significant increase of gene expression using equimolar amounts of either plasmid or Minicircle DNA.

Synthetic chemokine derived peptides for gene delivery via CXC receptor 4 Anna Egorova 1; Anton Kiselev 1; Vladislav Baranov 2; Arto Urtti 1 1University

of Helsinki, Faculty of Pharmacy, CDR, Helsinki, Finland; 2Ott’s Institute of Obstetrics and Gynecology, Lab. Prenatal Diagnostics, Saint-Petersburg, Russian Federation

Cell and tissue-specific DNA delivery can be achieved by derivatizing vehicles with a targeting ligand for certain receptor. CXCR4 is a receptor of chemokine SDF-1and is expressed on some types of cancer and stem cells. CXCR4 also interacts with the viral protein vMIP-II. The aim of this project is to characterize the group of CXCR4 targeted peptides for receptor-mediated gene delivery. We studied two peptides derived from SDF-1 and one peptide- from vMIP. SDF-1 peptides consist of N-terminal sequence of the chemokine SDF-1(residues 1-8). Additional binding motif (residues 12-17) was included into the longer peptide. Viral peptide was derived from N-terminal region of vMIP-II and consists of all D-amino acid analogs. All peptides are modified with DNA-binding sequence (K8). Control peptide consists of only K8 sequence. All peptides were able to condense DNA as shown by EtBr assay. Sizes and z-potentials of the DNA/peptides complexes were measured by means of a Malvern zeta/particle sizer. CXCR4 expression on A172, HeLa and CHO cell lines was estimated using flow cytometry. Peptides affinity to CXCR4 was proved by competitive displacement of monoclonal anti-CXCR4 antibody 12G5 from CXCR4() A172 cells. Transfectional efficacy of chemokine and vMIP derived peptide after glycerol treatment in CXCR4() HeLa and A172 cells was higher compared to control peptide. The level of transgene expression with signal peptides was comparable with the efficacy of control PEI. Signal peptides transfection efficacy on CXCR4(-) CHO cells was much lower than in control PEI. Intracellular uptake of signal peptides was more efficient than that of control oligolysine. We found that SDF-1 derived peptides were more potent than vMIP-II derived one. Thus we conclude that reported bifunctional, synthetic peptides represent an efficient DNA vectors for the CXCR4-targetd gene delivery. This work was supported by Galenos fellowship (Marie Curie Contract MEST-CT-2004-404992), INTAS fellowship 06-1000014-6073 and partly supported by CRDF grant ST012, RFBR grant 06-04-08338 and grant of administration of St.-Petersburg. E-mail: [email protected] P 144 Large scale minicircle-DNA production becomes reality Marco Schmeer 1; Markus Blaesen 1; Peter Mayrhofer 2; Wolfgang Jechlinger 2; Ruth Baier 3; Martin Schleef 4 1PlasmidFactory

GmbH & Co. KG, R&D, Bielefeld, Germany; & Jechlinger OEG, R&D, Vienna, Austria; 3PlasmidFactory GmbH & Co. KG, Production, R&D, Bielefeld, Germany; 4PlasmidFactory GmbH & Co. KG, CEO, Bielefeld, Germany 2Mayrhofer

Background: Plasmid DNA is commonly used in vaccination, gene- or cell therapy but also as a basic substance in viral vector production. The presence of antibiotic (AB) re-

E-mail: [email protected] P 145 Transduction of liver non-parenchymal cells by hydrodynamic tail vein injection of plasmid DNA Xavier M Anguela ; Sabrina Tafuro ; David Callejas ; Joel Montane ; Cristhopher J Mann ; Fatima Bosch Universitat Autònoma de Barcelona, Center of Animal Biotechnology and Gene Therapy, Edifici H - CBATEG, Bellaterra, Spain Hydrodynamic-based gene transfer is a well-established technique to transduce the liver as a way of treating hepatic disorders or for releasing proteins into the circulation. In mice, the technique involves the injection of an expression cassette in a volume of solution equal to 10% of body weight via the tail vein in less than five seconds. In larger animals, the technique requires local injection into the hepatic or portal vein and generation of pressure by occluding the hepatic circulation. All current studies in mice have shown that after hydrodynamic nucleic acid delivery only hepatocytes are transduced. However, these conclusions were based on histological studies. Our aim was to fully characterize the repertoire of transduced cells using both histology and more sensitive assays including flow cytometry and PCR. In contrast to other studies, we show here that in addition to hepatocytes, other hepatic cells were also transduced after the hy-

ESGCT 2008 POSTER PRESENTATIONS drodynamic injection of a plasmid encoding a reporter gene. Proportionately, the most transduced non-parenchymal cell type observed was Kupffer cells. We used RT-PCR analysis to confirm that these transduced cells were expressing the transgene to discard the possibility that the cells were phagocytosing the protein and not expressing it. In addition, other non-parenchymal cell types were also GFP-positive. We compared transduction efficiencies using several constructs, including those containing ubiquitous and hepatocyte-specific promoters, as well as comparing different doses. Importantly, since a significant percentage of non-parenchymal immune liver cells, such as Kupffer and dendritic cells, were transduced, our data may have implications for the interpretation of gene therapy experiments targeting the liver. These results may also help in the design of immunomodulatory therapies, particularly those targeting marcrophages for the treatment of inflammatory diseases. Supported by SAF2005-0162, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) and CLINIGENE. E-mail: [email protected]

P 146 Transfection ability and intracellular DNA pathway of nano-structured gene delivery systems Xin Zhang 1; Jean-Claude Voegel 1; Yves Mély 2; Nadia Benlirane-Jessel 1 1INSERM, Unité 595, Faculté de Médecine, Université Louis Pasteur, Strasbourg, France; 2Département de Pharmacologie Physiochimie, Faculté de Pharmacie, Université Louis Pasteur, Illkirch, France

Background: Considerable efforts have been devoted to the design of structured materials with functional properties. Polyelectrolyte multilayer films are now a well-established nanostructured concept with numerous potential applications, in particular as biomaterial coatings. This technique allows the preparation of nanostructured architectures exhibiting specific properties for cell-activation control and local drug delivery. Method: In this study, we used a multilayered system made of poly-(L-lysine)/hyaluronic acid (PLL/HA) as a reservoir for active DNA complexes with nonviral gene delivery vectors, PLL, beta-cyclodextrin (CD), and PLL-CD. The transfection efficiency of the DNA in complexes with PLL, CD and PLL-CD with the polyelectrolyte multilayers (PEM) were investigated by comparison of the luciferase gene expression of the complexes. The pDNA intracellular pathway were investigated by comparison of the luciferase gene expression of these complexes and an endocytosis marker FM 4-64 visualised by confocal microscopy. The some experiment was performed in solution. Results: When embedded into the multilayered films, the transfection efficiencies of the DNA complexes and the cell viability were improved. The highest transfection was obtained with the PLL-CD/pDNA complexes. We found this high transfection efficiency was related to an efficient internalization of the complexes in the cell cytoplasm and selected nuclei domain through a nonendocytotic pathway. Conclusion: For the fist time, we report the intracellular pathway of the pDNA in complexes incorporated into the multilayered system. Note: This work has been in press in Nano Letters. E-mail: [email protected]

1153 P 147 Folate-associated cationic liposomes as a promising system for tumor-targeted gene delivery Sónia Duarte ; Henrique Faneca ; Sara Trabulo ; Ana Cardoso ; Maria da Conceição Pedroso de Lima Center for Neuroscience and Cell Biology, Department of Biochemistry, University of Coimbra, Coimbra, Portugal Background: Cationic liposomes have several attractive features for gene transfer and have been extensively utilized for gene delivery, both in vitro and in vivo. However, their in vivo efficiency is still unsatisfactory, so it is urgent to optimize their performance by developing novel, efficient and targeted formulations. Lipoplexes can be rendered more selective for cancer cells by targeting them to receptors, such as folate receptor, that are overexpressed on these cells. Methods: In this work, we used lipoplexes prepared from liposomes composed of EPOPC:Cholesterol (Chol) or DOTAP:Chol in the presence of folate. Folate was either associated electrostatically or attached to liposomes via the PEG spacer arm. The latter strategy allows combining targetability and longevity of these systems, which is particularly important when cancer cells are inaccessible or there are multi-located tumor sites, under which conditions delivery systems must be administered systemically. We evaluated the efficiency of these systems to deliver a reporter gene to TSA (tumor mammary adenocarcinoma) cells in vitro, in the presence or absence of serum, by measuring luciferase activity, and cytotoxicity by Alamar Blue assay. Results: Our results show that electrostatic association of folate (up to 200g/g plasmid) potentiates the biological activity of both EPOPC and DOTAP systems. Such increase is further improved in the presence of 10% of serum, in the case of DOTAP-based systems but slightly decreased for EPOPC systems. Incorporation of PEG in liposomes decreases drastically the biological activity of the complexes. Nevertheless, when folate is covalently linked to PEG the biological activity of the complexes is slightly enhanced, this increase being potentiated upon concomitant association with folate. Conclusion: Overall, association of folate to the lipoplexes results in high levels of transfection activity and combination of this strategy with that involving coupling of folate to PEG may be of great relevance for future in vivo applications. E-mail: [email protected] P 148 Exploration into the structural diversity of arginine conjugated PAMAM dendrimers for efficient gene delivery systems ChengZhe Bai ; Tae-il Kim ; Kihoon Nam ; Jong-sang Park Seoul National University, Department of chemistry, seoul, Republic of Korea Background: Dendrimers are polymers with defined structures, inner cavities able to encapsulate guest molecules, and controllable multi-valent functionalities in their inner or outer part. Recently we reported that conjugation of arginines to PAMAM dendrimer G4 greatly enhanced their transfection efficiency. Therefore we intend to conjugate diarginines to the amines of the dendrimers and to vary the PAMAM dendrimer generation in order to examine the structure-activity relationships of arginine conjugation for the first time.

1154 Methods: PAMAM-R was synthesized by conjugating Fmoc-Arg(pbf)-OH to the surficial amines of PAMAM dendrimer. In the case of PAMAM-R2, after removal of the Fmoc groups of PAMAM-R, arginines were repeatedly conjugated to the primary amines of arginines residues grafted to PAMAM-R. Synthesized dendrimers were characterized by 1H NMR, agarose gel electrophoresis, size and zeta-potential measurements of polyplexes, polyplexes stability assay, cytotoxicity assay, in vitro transfection experiment and cellular trafficking observation. Results: Number of conjugated arginines were examined by 1H NMR (PAMAM3-R : 31, PAMAM4-R : 60, PAMAM3R2 : 59, PAMAM4-R2 : 116). They could retard pDNA at a charge ratio of 2 and form polyplexes with sizes less than 250nm from a charge ratio of 4. Di-arginine conjugated dendrimers showed higher Zeta-potential values and polyplex stability than mono-arginine conjugates. Their cytotoxicities were low but increased in accordance with the increase of charge densities and molecular weights. Except for PAMAM3-R, they displayed high transfection efficiency comparable to, or higher than PEI25k in mammalian cells. Diarginine conjugates showed greater efficiency than mono-arginine conjugates in the serum condition. PAMAM4-R2 polyplexes were observed to show the greatest intra-nuclear localization but this was not directly related to the highest transfection. Conclusion: Some degree of arginine conjugation brings high transfection, and that di-arginine conjugation to PAMAM dendrimers leads to great polyplex stability and intra-nuclear localization. However, a novel strategy for adjusting cytotoxicity and polyplex stability will be needed. E-mail: [email protected] P 149 Development of new eukaryotic expression vectors devoid of antibiotic resistance markers Corinne Marie 1; Mickael Quiviger 1; Magali Richard 1; Gaelle Vandermeulen 2; Veronique Preat 2; Daniel Scherman 1 1INSERM, CNRS, ENSCP, Universite Paris Descartes, Unite de Pharmacologie Chimique et Genetique, Paris, France; 2Universite Catholique de Louvain, Unite de Pharmacie Galenique, Brussels, Belgium

Nonviral Gene Therapy requires production of large quantities of plasmid DNA that should meet, for clinical trials, defined criteria such as biosecurity, tissue-specific gene expression as well as quality of pharmaceutical grade (e.g. supercoiled structure . . . ). Maintenance and propagation of nonviral plasmidic vectors, in bacterial cells, necessitate to exert a selection pressure. To avoid unnecessary dissemination of antibiotic resistance markers, we developed a new system to produce plasmids Free of Antibiotic Resistance genes, referred as pFARs. The strategy is based upon the suppression of a chromosomal nonsense mutation by a plasmid-borne function. The amber mutation was introduced into the thyA gene that encodes a thymidylate synthase involved in dTMP synthesis, resulting in thymidine auxotrophy. In parallel, a small plasmid vector, called pFAR4 carrying a suppressor t-RNA gene was entirely synthesised. Introduction of pFAR4 into the thyA mutant restored normal growth to the auxotrophic strain. Further optimisation of the bacterial producer strain, consisting of deleting both the endA and recA genes that encode an endonuclease affecting plasmid purity and a re-

ESGCT 2008 POSTER PRESENTATIONS combinase involved in multimers formation, respectively allowed to produce predominantly monomeric supercoiled plasmids, as required for clinical trials. Luciferase activities measured after injection and electrotransfer of pFAR4-CMV LUC BGH into mouse muscle were similar to those obtained with a commercial vector containing the same expression cassette. Furthermore, whereas with traditional expression vectors, luciferase activities progressively declined after injection into mouse skin, sustained levels were observed with pFAR derivatives. To prevent protein expression in antigen-presenting cells and stimulation of immune responses, a short muscle-specific promoter region, called MCK for Muscle Creatine Kinase (Fabre et al, 2006, J. Gene Med. 8:636-645) was identified and introduced into pFAR4. Thus, bacterial and plasmid optimization led to the development of a small biosafe eukaryotic expression vector for gene therapy. E-mail: [email protected] P 150 Development of cationic-fatty acids for the purpose of in vivo gene delivery Joana R. Viola 1; Hans Leijonmarck 2; Merita Murtola 2; Oscar Simonson 1; Roger Stromberg 2; C.I. Edvard Smith

1

1Karolinska Institutet, Laboratory Medicine, Stockholm, Sweden; 2Karolinska Institutet, Dep. of Biosciences and Nutrition, Stockholm, Sweden

Lack of efficient in vivo gene delivery is a well-known shortcoming of non-viral delivery vectors. We have developed a series of novel plasmid-based non-viral carriers for in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with amphiphilic synthetic derivatives of naturally occurring molecules. We have first tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA), by gel retardation assay, and shown the influence of the hydrophobic portion in the migration of the DNA nanocomplexes. Depending on the cationic component, DNA was found to be protected from DNase I degradation and this protection correlated with the length of the alkyl chain. Within the concentration range that was tested, most of these conjugates induced no, or low, cell toxicity, as shown by in vitro cytotoxicity studies. Further studies allowed the selection of the most promising candidates for in vivo delivery. In the skeletal muscle, DNA nanoparticles ensuing from two of these selected carriers resulted in a ten-fold increase of expressed protein as compared to the pDNA. Herein, we present a new class of cationic-fatty acid conjugates that have been shown to hold potential for an efficient in vivo gene delivery to skeletal muscle. E-mail: [email protected] P 151 Evaluation of cystein-flanked cross-linking peptides as carriers for gene delivery Anton Kiselev 1; Anna Egorova 1; Vladislav Baranov 2; Arto Urtti 1 1University

of Helsinki, Faculty of Pharmacy, CDR, Helsinki, Finland; 2Ott’s Institute of Obstetrics and Gynecology, Lab. Prenatal Diagnostics, Saint-Petersburg, Russian Federation Cross-linked gene delivery systems which are capable to trigger the intracellular release of DNA promise an im-

ESGCT 2008 POSTER PRESENTATIONS provement in the efficacy of DNA delivery process. Cysteinflanked peptides form small, stable DNA condensates because of cross-linking. Intracellular reducing environment can cleavage disulfide bonds in the DNA-polyplexes and it also decreases peptide toxicity. The aim of the project is to characterize the group of cross-linking peptides for gene delivery purposes. Two oligolysine (CHKKKKKKHC, CHKKKKKKHHKKKKKKHC) and one oligoarginine (CHRRRRRRHC) peptides were studied. The peptides modification with histidine residues facilitates the escape of DNA from lysosome. Two types of polymerization of cross-linking peptides were carried out. First type was performed during DNA/carrier complex formation (polymerization on DNA template). The second one was done before complex formation (oxidative polycondensation). EtBr exclusion and DNAse I protection assays proved peptides ability to condense and to protect DNA. DNA-polyplexes could be destabilized by DTT with DNA release. Template polymerization during DNA complexation was studied using continuous fluorescent assay with parallel measurement of free thiol groups in DNA-polyplexes. Time dependence between density of complexes and amount of free thiol groups was found. Transfection activity was studied in several cell lines (CHO and HeLa) with luc as a reporter gene. Transfectional analysis of template polymerized carriers showed more efficient transgene expression for oligoarginine peptide. A 100-1000fold higher level of gene expression for polymers with oxidative polycondensation was indicated in comparative transfection analysis of peptides with both types of polymerization. There was a 10-times higher level of luciferase activity using oxidatively polymerized oligoarginine compared with control PEI. Thus these results demonstrate that oxidative polycondensation of cross-linking peptides provides a perspective approach in development of effective non-viral gene delivery systems. This work was supported by Galenos fellowship (Marie Curie Contract MEST-CT-2004-404992), INTAS fellowship 06-1000014-6073 and partly supported by CRDF grant ST012 and RFBR grant 06-04-08338. E-mail: [email protected] P 152 Nanosecond electric pulses-induced enhancement of reporter gene expression after electrotransfer in skeletal muscle in vivo Julien Villemejane ; Vanessa Joubert ; Aude Silve ; Lluis M. Mir CNRS, UMR 8121 CNRS - I. Gustave-Roussy - U. Paris-Sud, Villejuif, France We explored whether electric pulses of a duration of a few tens of nanoseconds (nsPEF) and of very high electric field amplitude (20 to 150 kV/cm), called nanopulses, could affect the expression of electrotransferred genes, improving the overall efficacy of non-viral, electric pulses-mediated, gene transfer. Contrary to the micro- and milliseconds pulses, the nanopulses act on the cell inside and may have targets other than the cell plasma membrane. DNA coding for the luciferase was electrotransferred into skeletal muscle of mice using conventional procedures of electrotransfer. Then tissues were exposed or not to nanopulses (10 ns duration) with a new exposure technique. nsPEF were applied by using insulated electrodes in order to overcome electrochemical reactions that could interfere with biological material and to prevent direct contact with high electric voltage. Muscle were processed 48 or 72 hours

1155 after. Measurements were performed using a luminometer and results were expressed as pg luciferase/mg muscle. An increase of two to three times of the luciferase activity in the extracts can be achieved with the application of a train of 30,000 nanosecond pulses of 30 kV (corresponding to a 40kV/cm static field), 0 to 60 minutes after plasmid electrotransfer. The time between DNA electrotransfer and nanopulse(s) delivery (between 0 and 60 minutes) seems to affect biological results. Other parameters influencing the luciferase gene expression as well as the mechanisms underlying such increase are under analysis. Cells and tissue manipulation by means of electric nanopulses delivery might be an efficient way to increase the overall efficacy of gene electrotransfer. E-mail: [email protected] P 153 Development of sleeping beauty transposon system mediated gene transfer method for gene therapy Tytteli Turunen 1; Zsuzsanna Izsvák 2; Seppo YläHerttuala 1 1University of Kuopio, A. I. Virtanen Institute for Molecular Sciences, Kuopio, Finland; 2Max Delbrück Center for Molecular Medicine, Berlin, Germany

Transposon vectors are non-viral gene transfer vehicles generated from natural mobile DNA-sequences, transposons. As transposons integrate themselves into chromosomes as they move in genome, also the gene transfer mediated by transposon vector evokes integration and stable expression of a therapeutic gene. In an inherited autosomal disease of cholesterol metabolism, familial hypercholesterolemia, lack of functional LDL receptors (LDLR) leads to strikingly elevated plasma LDLcholesterol levels and premature atherosclerosis. This study is based on Sleeping Beauty (SB) transposon system’s ability to integrate the therapeutic LDL receptor gene into the genome of the LDLR defective cells. Traditionally SB transposon system consists of two components: a transposon plasmid, which carries a transgene cassette, and a helper plasmid, which encodes for transposase protein that mediates the transposition reaction of the transposon into the genome. Since the optimal transposase to transposon (Ts:Tn) ratio for HepG2 human hepatoma cell culture was defined by G418 counterselection of NeoR-positive cell colonies to be unexpectedly 1:1, helper independent transposon-transposase plasmids were constructed and further cell culture experiments were conducted. Our initial results suggest that the helper independent system performs at least comparably to the traditional two component system with Ts:Tn ratio 1:10 in HepG2 culture. Presently we are transferring lacZ marker gene in both the helper independent and the two component transposon systems into LDLR deficient mice to determine the optimal Ts:Tn ratio for in vivo gene transfer. We will proceed by transfecting the mice with therapeutic LDLR transposon constructs to see the constructs’ ability to reduce plasma LDLcholesterol concentration and atherosclerosis in the animals. E-mail: [email protected] P 154 Successful transfection of human air liquid interface cultures in vivo with the non-viral gene transfer agent GL67A Uta Griesenbach 1; Catarina C Vicente 1; Ann-Marie Green 2; Seng H Cheng 3; Ronald K Scheule 3; Ian A Pringle 4; Deborah R Gill 4; Stephen C Hyde 4; Eric WFW Alton 1

1156 1Imperial

College London, Department of Gene Therapy, UK CF Gene Therapy Consortium, London, United Kingdom; 2University of Oxford, Gene Medicine Group, UK CF Gene Therapy Consortium, Oxford, United Kingdom; 3Genzyme Corporation, Genzyme, Framingham, United States; 4University of Oxford, Gene Medicine Group, UK CF Gene Therapy Consortium, Oxford, United Kingdom Differentiated human air liquid interface (ALI) cultures recapitulate properties of human airway epithelium. However, ALIs are notoriously difficult to transfect and, to the best of our knowledge non-viral gene transfer has not been successfully demonstrated in this model. We, therefore, assessed cationic GL67A-mediated gene transfer in fully differentiated ALIs purchased from Epithelix. We first complexed GL67A to a plasmid carrying the commonly used firefly luciferase (lux) reporter gene under the control of a CMV promoter (pCIK-FLux). ALIs were transfected using various amounts of DNA (2 and 10 g), DNA:lipid ratios and transfection times (6 and 24 hr). 48 hr after transfection cell lysates were prepared, but we were unable to detect lux expression in any of the samples. Recently a novel secreted luciferase reporter gene isolated from Gaussia princeps (GLux) has been described (Tannous 2005). We next transfected ALIs with GL67A complexed to a plasmid carrying secreted GLux under the control of a CMV promoter (pCMV-GLux). ALIs were transfected using 10 g pCMV-GLux/insert complexed to GL67A either at a 1:4 molar ratio (previously optimised for mouse nasal instillations) or at a 6:8 molar ratio (previously optimised for clinical trials). 48 hr after transfection apical secretion of GLux was quantified by washing the apical surface. Importantly, significant levels of GLux were secreted apically and basolaterally in ALIs transfected for 6 hr (1:4 ratio: 1.0  103  8.8x102 RLU/ul, 6:8 ratio: 1.3  104  1.1  104 RLU/ul, control: 2.1  102  41.5 RLU/ul, p  0.05, n  6/group), but not in ALIs transfected for 24 hr. Interestingly, the lipid:DNA ratio previously optimised for human lung transfection (6:8 molar ratio) lead to higher (p  0.05) transfection than the 1:4 molar ratio. Similar to in vivo models CMV-mediated expression fell to baseline levels within 1 week after transfection. In conclusion: (1) secreted Gaussia luciferase may be a more sensitive reporter gene than firefly luciferase, (2) GL67A transfects fully differentiated human ALIs, (3) gene expression can be followed over time by repeated washing of the apical cell surface. E-mail: [email protected] P 155 Impact of polymers on lipid cationic mediated gene transfer Raphael Chevre 1; Emilie Letrou-Bonneval 1; Romain Labas 1; Benoit Barteau 1; Olivier Lambert 2; Bruno Pitard 1 1INSERM, U915, Nantes, France; 2CNRS, CBMN UMRCNRS 5248 IECB, Talence, France

Background: Amphiphilic block copolymers, a new family of non toxic and FDA approved molecules, have recently been developed and displayed very efficient in vivo transfection activity in various tissues including skeletal muscle, lung and heart. However their mechanism remains unknown and better understanding of this mechanism represents a major goal for the future development of this new class of vectors. Methods: Surprisingly, we observed that lutrol, an amphiphilic block copolymer family member does not allow to

ESGCT 2008 POSTER PRESENTATIONS transfect in vitro cultured cells, suggesting that cell environment (in vitro or in vivo) is strongly involved in their mechanism of action. However electron microscopic examination of cells transfected with DNA formulated with block copolymer indicated that DNA molecules were internalized. The present study aimed at understanding this discrepancy between in vitro and in vivo transfection behaviour of block copolymers. Results: We observed that cell incubation with amphiphilic block copolymers prior cationic lipid mediated transfection improves the reporter gene expression, compared to that obtained without pre-incubation with block copolymer. Among the various steps going from cell internalization to transcription and traduction that could be improved by the block copolymer we found that the DNA molecules internalization step is probably the most important barrier on which the block copolymer played. Conclusion: Our results support that amphiphilic block copolymers improve transfection efficiency by allowing a better crossing of the cell membrane barrier both in vitro or in vivo. However even if the mode of entry of DNA in the presence of block copolymer in cells is probably the same, the transfection efficiency is very different. It is generally accepted that cells cultured in vitro mostly internalize DNA by clathrin dependent and independent mechanism requiring cationic lipid to escape the endosomes. DNA molecules do not probably follow this pathway of internalization in vivo and thus do not required cationic lipids to escape from vesicles. Amphiphilic blocks copolymers allow to cross efficiently the cell membranes but do not facilitate endosomal escape suggesting a direct delivery in the cytoplasm. E-mail: [email protected] P 156 Development of novel crosslinked poly(ethylenimine) as potential gene transfer agent King Sun Siu ; Marie Chia-Mi Lin The University of Hong Kong, Chemistry, 8N04 Kadoorie Building, Pokfulam Rood, Hong Kong, Hong Kong Background: Poly(ethylenimine) (PEI) has been considered as a potential gene delivery agent, however, it suffers from high cytotoxicity and poor in vivo transfection efficacy. Many researchers focused on giving PEI new features such as conjugating targeting ligands or crosslinking low molecular weight PEI (Mw 800, Mw 1300 and Mw 2000) or oligomeric PEI (Mn 423) by degradable disulphide bonds or ester bonds. However, the size or carbon numbers in the crosslinkers were arbitrary. Little is known in relating size or carbon number in crosslinkers respect to transfection efficacy and cytotoxicity. Methods: We hypothesize that optimizing the crosslinker size, hydrophobicity and charge density of PEI may result in reduced cytotoxicity, increased transfection efficiency and improved biocompatibility. We have designed and synthesized a series of crosslinked polymer for gene delivery by conjugating PEI Mw 800 to a biocompatible linker. The amide bonds of the crosslinked polymer were susceptible to be degraded slowly by hydrolysis and the degraded products are then readily removed from body. Results: The crosslinked PEI were tested for transfection efficacies in mammalian cell lines. Plasmid DNA encoding Enhanced Green Fluorescence Protein (EGFP) was used as a reporter gene. The protein expression was examined by fluorescence microscope. Cyctotoxicities of the crosslinked PEI were determined by MTT assay. These crosslinked PEI have

ESGCT 2008 POSTER PRESENTATIONS higher in vitro transfection efficacies but reduced cytoxicities as compared to PEIw 25k in 293A, U373, U87, B16 and HepG2. Conclusion: Further optimizations of these crosslinked PEI should provide a better non-viral vector for gene delivery system. E-mail: [email protected] P 157 Targeted -cyclodextrine-based gene delivery systems: Physicochemical and transfection studies Nicolas Guilloteau 1; Loic Legourrierec 1; Alejandro DiazMoscoso 2; Carmen Ortiz Mellet 3; Juan M. Benito 2; Christophe Di Giorgio 1; Pierre Vierling 1; Jacques Defaye 4; José M. García Fernandez 2 1LCMBA-UMR6001-CNRS,

Université de Nice Sophia Antipolis, Nice, France; 2Instituto de Investigaciones Químicas, CSIC, Sevilla, Spain; 3Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, Sevilla, Spain; 4Institut de Chimie Moléculaire de Grenoble, Dépt. de Pharmacochimie Moléculaire, Grenoble, France Background: Synthetic carriers that could prove as efficient as viral gene delivery systems but safer to use, homogeneous and non-immunogenic represent the ultimate challenge for gene therapy. Cyclodextrines (CDs), as nonimmunogenic and biodegradable biomaterials with high inclusion capabilities, present specific and interesting features for the development of artificial viruses. In this context, we developed amphiphilic cationic -CD derivatives with a defined architecture to perform specific tasks through the assembly of programmed building blocks. Unprecedented nonspecific gene transfection efficiencies and low toxicities for CDplexes (complexes formed between the CDs and DNA) have been achieved by the incorporation of thiourea segments, a dendritic display of amino groups, and a cluster of short lipophilic chains onto the CD platform. Methods: In order to increase (i) the cell recognition of the CDplexes by specific ligand-receptor interactions, and (ii) more particularly their circulation time in the blood for in vivo gene delivery, new saccharidic-labelled CD derivatives and ligandbased conjugates for inclusion into the CDs have been designed for the formulation of targeted CDplexes. This has been achieved by substitution of the terminal amino groups by saccharidic clusters, or using the capability of inclusion of adamantane into the cavity of the CD with the elaboration of an adamantane-PEG-folic acid conjugate. Results and conclusion: The saccharidic CDs did efficiently compact pDNA into stable, 100-200 nm-sized CDplexes. Mannose-labelled CDplexes were found in vitro to transfect specifically COS-7 cells which express mannose receptors. Post-labelling of cationic CDplexes by AD-PEG-folic acid (inclusion into the cavity of the CDs) was confirmed by zeta potential. For these targeted systems, transfection assays with folate-overexpressing KB cells are under progress. E-mail: [email protected] P 158 DNA electrotransfer into solid tumors in mice by combination of high- and low-voltage electric pulses

Maja Cemazar 1; Muriel Golzio 2; Pernille Hojman 3; Simona Kranjc 1; Suzana Mesojednik 1; Justin Teissie 2; Gregor Sersa 1

1157 1Institute

of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia; 2UMR 5089, IPBS-CRNS, Equipe de Biophysique Cellulaire, Toulouse, France; 3Rigshospitalet, Center of Inflammation and Metabolism, Copenhagen, Denmark Electrotransfer (electroporation) is recognized as one of the most promising alternative method to viral vectors for transfection of different tissues in vivo for therapeutic purposes. Recently, an increase in transfection efficiency was obtained in skeletal muscle and skin with a combination of short high voltage (HV) and long low voltage (LV) electric pulses compared to train of identical pulses. The aim of our study was to evaluate the transfection efficiency of reporter genes in subcutaneous tumors using different combinations of HV (600-1300 V/cm, 100 s, 8 pulses) and LV (80-160 V/cm, 50-400 ms, 1-8 pulses) pulses and to compare it to standard protocol using 8 identical pulses of 600 V/cm and 5 ms duration (EGT). B16 melanoma and SA-1 fibrosarcoma murine tumors were used. They were treated 6 - 8 days post-transplantation with intratumoral injection of 50 g of either pEGFP-N1 coding for green fluorescent protein (GFP) or pCMV-Luc plasmid coding for luciferase and immediately thereafter exposed to electric pulses, which were generated by CLINIPORATOR™ and Jouan GHT. Expression of GFP was determined on whole tumors ex vivo and on frozen tissues sections using fluorescence microscope and expression of luciferase by measuring its activity using luminometer. In both tumor models, the standard EGT protocol yielded the highest expression of both reporter genes. However, certain combinations of HV and LV pulses can result in similar transfection efficiency as EGT pulses. In combination electroporation protocol, relatively high voltages of LV pulses were necessary to obtain comparable transfection to standard protocol. Expression of reporter genes was higher in B16 melanoma compared to SA-1 fibrosarcoma. Our results proved the feasibility of the combination of HV and LV electric pulses for delivery of plasmid DNA to tumors in vivo. However, further studies on the underlying mechanisms involved in the electrotransfer to tumors and detrimental effects on normal tissues should be performed in order to foster electrogene therapy into the clinical trials. This work was supported by the ARC, the Ligue contre le Cancer de Midi Pyrenees, the Region Midi Pyrenees, the AFM, the EU project Cliniporator, a Slovenian CNRS PICS and Slovenian Research Agency (P3-0003, J3-7044). E-mail: [email protected] P 159 Ultrasound-mediated gene transfer (sonoporation) and expression in vivo Ying Suet Li ; Nicolitsa Nomikou ; Anthony P McHale University of Ulster, School of Biomedical Sciences, Cromore Rd., Coleraine, Co. Derry, United Kingdom Background: The use of ultrasound to facilitate transient and non-destructive cell membrane poration (sonoporation) represents a relatively novel means of introducing nucleic acid-based medicines into cellular and tissue-based targets. The objective of the work presented here is to illustrate the wide-ranging potential of such an approach by demonstrating functional gene expression in vivo using a variety of tissues/target sites. Methodology: Ultrasound-mediated gene transfer (sonoporation) was performed in C3Hen mice using an SP100

1158 sonoporator together with the MB101 microbubble (Sonidel Ltd.). Luciferase-encoding plasmids, pCMV-Luc and pEPI1-Luc (PlasmidFactory GmbH & Co) were employed and gene expression was detected by bioluminescent imaging using the Xenogen IVIS system. Results: In this study, ultrasound-mediated gene transfer into both muscle and tumour tissues (RIF-1) were examined in vivo. The data demonstrated that ultrasound facilitated gene transfer in both tissues and in particular cases, luciferase expression could be detected up to 200 days following the gene transfer event. Interestingly, gene expression could also be detected in tumour tissues throughout the lifetime of the host animal and the bioluminescent signal increased with increasing tumour size. Conclusions: Ultrasound may be employed to facilitate long-term gene expression in vivo and based on our data, the results suggest that this approach may be employed in gene therapies directed towards the treatment of cancer. E-mail: [email protected] P 160 Development of non-viral gene delivery systems based on poly-L-glutamic acid and poly-L-arginine derivatives Vincent Vermeersch 1; Veska Toncheva 1; Sandra Van Vlierberghe 1; Lorenzo Tibaldi 2; Alain Joliot 2; Peter Dubruel 1; Etienne Schacht 1 1Ghent

University, Polymer Chemistry & Biomaterials Research Group, Krijgslaan 281, S4bis, Gent, Belgium; 2Ecole Nationale Supérieure (ENS), Département Biology, Rue d’Ulm, Paris, France Background Since the current treatments for age related macula degeneration are not adequate enough, there is an obvious need for improved therapy. The use of non-viral gene delivery systems might be one of the possible solutions. In the present work, we report on the development of biodegradable cationic polymers to be applied as delivery system for therapeutic DNA. Method & Results Multifunctional cationic polymers derived from poly--amino acids (L-glutamic acid and L-arginine) were obtained by polymerisation of their N-carboxyanhydrid derivatives and further modified to obtain the desired property. The formation and behaviour of inter-polyelectrolyte complexes (IPEC) between oppositely charged DNA and polymers was investigated using several charachterisation methods and compared to polyethyleneimine (PEI). The biological properties were examined by transfection and toxicity studies and by confocal microscopy on ARPE19 cell lines using the RucHA model plasmid. Several polymers of different molecular weight (10–250 kDa) were synthesised. The composition of the synthetic vectors was varied to obtain side-groups containing primary amines, tertiary amines, imidazole, hydroxyl and guanidine functional groups linked to a backbone of poly-l-glutamine, poly-l-arginine or a combination of both. Part of the characterisation was carried out by determining the condensing capacity, stability and size(distribution) of polymer-DNA complexes using agarose gel-electrophoresis, ethidiumbromide fluorescence, dynamic light scattering and zeta-potential measurements. It was found that all polymers were able to form complexes at charge ratio’s (polymer/DNA) ranging from 0.52/1 and that the complexes are stable up to 7 days. All polyplexes tested were smaller than 100 nm (at CR 4/1). Biological tests (transfection efficiency) indicated that certain polymers (10-35% guanidine, 50% hydroxyl) achieved transfection levels comparable to those of PEI.

ESGCT 2008 POSTER PRESENTATIONS Conclusion It can be concluded that polymers based on poly-l-glutamic acid and poly-l-arginine with specific sidegroup functionalities are promising as novel synthetic vectors for gene delivery. Acknowledgment The authors would like to acknowledge the EC funding (EU-STREP, PolExGene, project 019114). E-mail: [email protected] P 161 Characterization and in vitro silencing effect of chitosan/siRNA complexes Emine Salva ; Julide Akbuga Marmara University, Pharmaceutical Biotechnology, Istanbul, Turkey Background: RNA interference (RNAi) by small interfering RNA (siRNA) is now being evaluated in cell culture and in vivo as novel therapeutic strategy. Potent gene inhibition by siRNA has been limited due to biological stability, extracellular and intracellular barriers. The efficiency of carrier system or delivery is an important subject in this technology. The objective of this study was to investigate the carrier ability of chitosan by the complexation of siRNA with chitosan, transfection efficiency and its silencing effect in vitro. Furthermore, we compared gene inhibition activities of siRNA targeting against beta-gal gene and plasmid DNA encoding anti-beta gal siRNA. Method: Chitosan/siRNA (CS/siRNA) and chitosan/ psiRNA (CS/psiRNA) complexes were prepared by using different concentrations of chitosan and siRNA. Full complexation were obtained at the ratio of 0.5/1 for CS/psiRNA and 5/1 for CS/siRNA. The in vitro characterization (size and charge) was made and silencing activity of appropriate formulations were investigated in vitro. The silencing activity was measured based on the inhibition of beta-galactosidase coded by pSV-beta-Gal plasmid followed by the addition of siRNA containing formulations into the HEK293 cells. ONPG method was used for the determination of beta gal activity. Results: Chitosan shows great potential for in vitro efficacy of siRNA. CS/psiRNA complexes (1/1-8/1) and CS/siRNA (15/1-5/1) have inhibited the beta-gal expression in different percentage (40-70%). There is no difference between the silencing activity siRNA and plasmid encoding siRNA. Time dependent and dose dependent silencing effect was observed. Conclusion: As a result, chitosan can be considered as a suitable carrier especially for siRNA. This study can form the basis for the forthcoming studies related with carrier systems of siRNAs that will be done with chitosan polymer. This study was supported by Marmara University Scientific Research Projects Association (BAPKO). E-mail: [email protected] P 162 Corona ions based applicator for mediating gene delivery Richard Gilbert 1; Mark Jaroszeski 1; Reyes Jose 1; Richard Connolly 1; Niraj Ramachandran 1; Andrew Hoff 2; Eric Pressen 3 1University

of South Florida, Chemical Engineering, Tampa, United States; 2University of South Florida, Electrical Engineering, Tampa, United States; 3University of South Florida, Center for Molecular Delivery, Tampa, United States

ESGCT 2008 POSTER PRESENTATIONS Electric field mediated drug and gene delivery are attractive methods for non-viral delivery of therapeutic molecules. Electropermeabilization (electroporation) using rectangular pulses and exponential wave forms is a recognized procedure to generate a mediating electric field. This poster presents a novel non-contact alternate procedure that employs corona ions to generate a force field to mediate delivery of a reporter plasmid. Results characterize and demonstrate the method’s potential in vitro and in vivo. Low energy corona ions generated at atmospheric pressure are directed toward cells in vitro, or animal tissues to mediate their respective environments to assist in targeted molecule delivery. Results presented summarize the applicator development using sytox green, and a chemotherapeutic agent, bleomycin, on B16F10 cells. Additional development indicated that bleomycin and corona ion application to solid tumors in animal models demonstrate a significant decrease in tumor growth rates in established aggressive murine melanomas. Further, the repeatedly observed region of effective drug delivery in all in vitro treated cells confirmed predicted electrostatic model expectations. Finally, studies are presented that show that a combination protocol including an intradermal injection of plasmid plus corona ion exposure results in successful protein expression E-mail: [email protected] P 163 Design and in vitro characterization of Tat derived cellpenetrating peptides Astrid Subrizi ; Maxim Antopolsky ; Arto Urtti University of Helsinki, Centre for Drug Research (CDR), Faculty of Pharmacy, Helsinki, Finland Background: Difficult transportation across the cell plasma membrane is a notorious hallmark for the intracellular delivery of therapeutic macromolecules, such as genetic material. Among the so-called cell-penetrating peptides (CPPs), Tat peptide was reported to be rapidly taken up by cells in culture and has been used for the delivery of various nanoparticulates. The aim of this study is to investigate the relationship between structure and activity of Tat peptide and to clarify its possible mechanism of cellular uptake. Methods: A series of sixteen peptides, homologue to the original Tat sequence, was synthesised. Modifications consisted in lipophilic, aromatic, neutral or non-natural amino acid residues, located in different areas of the original sequence, mostly between arginine residues of the native peptide. To study membrane permeation, the peptides were mixed with calcein-loaded liposomes with a lipid composition mimicking the membrane of cells. Permeation of the peptides across the model membrane was indirectly measured using a calcein dequenching assay. Cellular uptake and cytotoxicity of the peptides were studied at two different peptide concentrations in three cell lines by flow cytometry analysis. Intracellular localization and trafficking were visualised by confocal laser scanning microscopy (CLSM). Results: The permeation of Tat derived peptides across model lipid bilayers followed either “slow” or “fast” kinetics, a feature which may be relevant in predicting CPP’s ability to penetrate plasma membranes. CLSM allowed us to distinguish peptides possibly adhering to cell surfaces from peptides inside cells. All peptides showed preferentially punctual distribution inside cells indicating their presence in vesicular structures. None of the peptides could be detected into the nuclei. Quantitative assessment of peptide internalization was achieved by flow cytometry analysis.

1159 Conclusion: An attempt has been made to find chemical descriptors for cell penetration. Our study demonstrates that the sequence of Tat peptide can be modified without losing cell penetrating efficacy and, in certain cases, even increasing it. E-mail: [email protected] P 164 Altered activity of CMV promoter in stably transfected murine tumors cells by stress and demethylation Urska Kamensek ; Gregor Tevz ; Simona Kranjc ; Suzana Mesojednik ; Gregor Sersa ; Maja Cemazar Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia Background:The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in mammalian cells. Although is often thought of as a strong constitutive and unregulated promoter, its activity depends on host-cell transcriptional environment which is altered in a stressed cell, and is also susceptible to transcriptional inactivation due to several mechanisms, including DNA methylation. The aim of this study was to examine the influence of DNA methylation and stresses like irradiation and treatment with chemotherapeutic agent on the activity of the CMV promoter. Methods: For this purpose two cell lines LPB and TSA were transfected with CMV promoter driven green fluorescent protein (GFP) reporter gene (pCMV-EGFP) using electroporation. After 2 months of culturing under increasing concentrations of selection agent geneticin, resistant cells were obtained and clones with highest GFP expression were identified by fluorescence microscopy and isolated for subsequent experiments. To test the influence of DNA methylation on CMV inactivation, cells were first passaged for 1 month to allow for the methylation of DNA and then treated with DNA methylation inhibitor 5-azacytidine (1M). To test stress-induced regulation of the CMV promoter, cells were exposed to irradiation (6 Gy) and chemotherapeutic agent cisplatin (3g/ml). Three days after the treatments expression of reporter gene was determined using fluorescence microplate reader and fluorescence microscopy. Results: Treatment with 5-azacytidine partially reactivated the CMV promoter indicating that the inactivation was at least partially caused by methylation. Furthermore, exposure of cells to irradiation or cisplatin resulted in significant upregulation of the expression in both cell lines, although the effects were more evident in TSA cell line. Conclusion:Observed alternations in the activity of the CMV promoter limit the usefulness of this widely used promoter as a constitutive promoter. Therefore, the choice of promoter linked to the gene of interest is crucial for success of gene therapy as well as basic research. E-mail: [email protected] P 165 Listeria monocytogenes as vector for gene transfer into human brain tumor cells Ulf Nestler 1; Sebastian Rollnik 1; Chris Schulz 1; Trinad Chakraborty 2; Eugen Domann 2; Torsten Hain 2

1160

ESGCT 2008 POSTER PRESENTATIONS

1Justus

Liebig University, Neurosurgery, Klinikstrasse 29, Giessen, Germany; 2Justus Liebig University, Institute for Medical Microbiology, Frankfurter Strasse 107, Giessen, Germany Background: The facultative intracellular bacterium Listeria monocytogenes has been used for vaccination studies and has been described as gene transfer vector in several mammalian cell types. In this study we examined (1) whether and to which extent Listeria monocytogenes invades cultured cells of brain tumors, such as glioblastoma, glioma, meningioma and metastases and (2) whether expression of foreign genes in glioblastoma cells can be achieved. Methods: We tested listerial vectors on commercial cell lines U87, A172, U373 and on 27 cell lines derived from culturing tumor specimen obtained during neurosurgery. Internalization rates of five mutant bacterial strains, partly with plasmid based re-complementation of the internalin A/B operon were compared to the wild-type EGD-e. In 9 glioblastoma cell lines the plasmid based transfer of foreign genes lacZ and EGFP was studied in detail and transfection efficiencies were determined. Results: Deletion of the internalin operon decreased the invasion of bacteriae into brain tumor cells. Plasmid based overexpression of distinct internalins could increase internalization rates to values above the wild type rate. In glioblastoma, the pathogenic strain 4bL312 achieved foreign gene expression efficiencies up to 5 %. Conclusion: Listerial internalin B influences the uptake of Listeriae into neuroepithelial cells, whereas internalin A is more important for invasion of meningioma cells. It remains to be determined, whether transfection efficiencies will be suitable for therapeutic approaches, such as suicide gene therapy. Our ongoing research focuses on the development of suicide gene vector plasmids and on the use of attenuated bacterial strains. Plasmid based overexpression of bacterial surface proteins may increase bacterial uptake rates and help in selective targeting of neuroepithelial cells. E-mail: [email protected] P 166 Anisamide-targeted cyclodextrin nanoparticles for siRNA delivery in cancer cells: physicochemical characterisation and in vitro evaluation Jianfeng Guo 1; Raphael Darcy 2; Caitriona O’Driscoll

1

1University

College Cork, School of Pharmacy, College Road, Cork, Ireland; 2University College Dublin, Centre for Synthesis and Chemical Biology, Conway Institute, Dublin, Ireland Background: Modified cyclodextrins (CDs) have been shown to form vesicles and nanoparticles (1) with potential to encapsulate siRNA thereby providing protection from enzymatic degradation. siRNA can regulate gene silencing in mammalian cells through the RNAi pathway. Prostate carcinoma cells are known to overexpress the sigma receptor capable of being targeted by anisamide (AA) (2). We are initially investigating the physicochemical characterisation and in vitro evaluation of an anisamide-targeted stealth cyclodextrin (CD-AA). Methods: CD-AA was synthesized and the structure was verified using TLC and NMR. Particle size and zeta potential were characterised using DLS. Formation of vesicles was assessed by TEM and encapsulation of fluorescein; gel filtration was used to separate free and encapsulated dyes. The serum stability of CD-AA:siRNA particles was evaluated by

quantifying the turbidity at 630nm. The serum stability of complexed siRNA was investigated using gel electrophoresis. Cellular uptake in human prostate cancer cells (PC-3) was studied by flow cytometry. The gene silencing was verified by co-transfection with jetPEI•plasmid pGL3 DNA and CD-AA•GL3 luciferase siRNA. Luciferase activities were determined as RLU/g of protein at 24hr. Cytotoxicity was measured by MTT. Results: Particle sizes of CD-AA nanoparticles and CD-AA:siRNA complexes at various mass ratios (MR5, 10, 15, and 20, MR  mass CD:mass siRNA) were 200nm, with zeta potentials range 0mV to  40mV. TEM and gel filtration confirmed the formation of vesicles. Aggregation occurred at higher concentrations of CD-AA. Complexed siRNA remained stable up to 12hr. While cellular toxicity was low, cellular uptake and transfection did not occur. Conclusions: A nanoscale-size, targeted amphiphilic cyclodextrin has been developed, with an acceptable safety, which forms vesicles. Further modification is required to improve serum stability and transfection efficiency. 1. Bart Jan Ravoo, Raphael Darcy. Angew. Chem. 2000; 112, Nr. 23: 4494-4495. 2. Rajkumar Banerjee et al. Int. J. Cancer. 2004; 112:693-700. E-mail: [email protected] P 167 Enhancement of the expression of reporter genes electrotransferred in cells in culture by nanosecond electric pulses Vanessa Joubert ; Julien Villemejane ; Lluis M. Mir CNRS, UMR 8121 CNRS - I. Gustave-Roussy - U. Paris-Sud, Villejuif, France We explored whether electric pulses of a duration of a few tens of nanoseconds and of very high electric field amplitude (20 to 150 kV/cm), called nanopulses, could affect the expression of electrotransferred genes, improving the overall efficacy of non-viral, electric pulses-mediated, gene transfer. Contrary to the micro- and milliseconds pulses, the nanopulses act on the cell inside and may have targets other than the cell plasma membrane. DNA coding for the luciferase was electrotransferred into cells in culture using classical procedures. Then cells were exposed or not to nanopulses (10 ns duration), and incubated for 24 to 48 hours. Measurements were performed using a luminometer and results were expressed as pg luciferase/g protein. An increase of three times of the luciferase activity in the extracts can be achieved with the application of just 1 nanosecond pulse of 60 kV/cm, 60 minutes after plasmid electrotransfer, in an electroporation cuvette with 1 mm of gap between the electrodes. The time between DNA electrotransfer and nanopulse(s) delivery (between 15 and 180 minutes) seems to be not relevant which seems to indicate that the effect of the nanopulses is not at the level of the DNA uptake. More than one pulse can also be applied, as no loss of viability is associated to the exposure to a few hundreds of these nanopulses. The effects are almost independent of the nanopulses repetition frequency, but dependent of the amplitude of the nanopulse electric field. Mechanisms are under analysis, as cell manipulation by means of electric nanopulses delivery might be an efficient way to increase the overall efficacy of gene electrotransfer. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 168 Lack of inflammatory response upon systemic delivery of siRNA or DNA mediated by polyethylenimine (PEI) Anne-Laure Bolcato-Bellemin ; Marie-Elise Bonnet ; Patrick Erbacher Polyplus-Transfection, R&D, Bd S. Brant, ILLKIRCH, France Treatment of various diseases such as cancer, inflammatory or infectious diseases by nucleic acid therapy remains one of the main challenges for the next decade. To date, two main strategies are being developed. The first one consists in delivering plasmid encoding genes, which either restore the function of a deficient gene or kill a specific cell type, such as cancer cells. The second approach exploits the mechanism of RNA interference whereby siRNAs are delivered to inhibit a specific gene function. The success of these therapies depends upon delivery vehicles, which are able to selectively and efficiently deliver therapeutic nucleic acids to target organs with minimal toxicity [Ross et al., 1996]. To date linear polyethylenimine (L-PEI) has being widely used for in vivo delivery of nucleic acid due to its versatility and efficiency. The factors enhancing transfection efficiency of nucleic acid such as DNA or siRNA with PEI have been well-studied. However, when moving towards the clinic, it is crucial to identify potential side-effects induced by this delivery system. For this purpose, we have analyzed production of pro-inflammatory cytokines (TNF-a, IFN-b, IL-6, IL-12/IL-23 and IFN-b) and hepatic enzyme levels (ALAT, ASAT, LDH and ALP) in the blood after systemic injection in mice of DNA or siRNA delivered with L-PEI. Our data showed no major production of pro-inflammatory cytokines or hepatic enzymes after injection of DNA or siRNA complexed with PEI, making this molecule a delivery reagent of choice for nucleic acid therapeutics. E-mail: [email protected] P 169 Gene therapy targeting antigen presenting cells as an arthritis treatment in collagen type II induced arthritis Tove Eneljung ; Louise Henningsson ; Inger Gjertsson University of Gothenburg, Dept of Rheumathology and Inflammatory Research, 413 46 Gothenburg, Sweden Background: Rheumatoid arthritis (RA) is a chronic and destructive joint disease. During the last decade more efficient treatments, e.g. specific cytokine inhibitors, have been available. However these drugs are built on the principal of immunosuppression, which occasionally has side effects, such as severe infections. An ideal remedy for autoimmune diseases would be antigen specific tolerance, i.e. the pathological immune response is diverted from destruction of the host’s own tissues to its normal non-responsive state. We have earlier shown that prophylactic treatment with a lentiviral vector encoding the immunodominant epitope of collagen type II (CII) in a mouse model of RA, CII induced arthritis (CIA), significantly ameliorates the course of arthritis in an antigen specific manner. The aim of this study is to use the same system to treat the animals after arthritis induction. Methods: The immunodominant epitope of CII (amino acids 259-270) was inserted in the CLIP position of the Invariant chain (Ii) which enables endosomal targeting and expression on MHC class II on antigen presenting cells. As a

1161 control the original Ii with CLIP was used. The constructs were driven by an SFFV promoter and cloned into second generation lentiviral vectors. At day 2 after CIA induction, 5 000 000 viral particles/mouse were injected intravenously (iv) into the treatment group or control group respectively. Results: Mice treated with the lentiviral vector expressing the CII epitope aa 259-270 displayed a significantly less severe (p  0.04) and frequent arthritis compared to controls. No differences were found in the levels of CII specific antibodies. Conclusion: Tolerance induction using gene therapy with lentviral vectors encoding the immunodominant epitope in CII as an arthritis treatment significantly ameliorates the arthritis development in CIA. Further studies are ongoing to elucidate the timing, the amount of virus needed and mechanisms behind the findings. E-mail: [email protected] P 170 Amelioration of experimental arthritis by telomerasedependent oncolytic adenovirus that targets synovial fibroblasts Chao-Liang Wu 1; Shin-Yao Chen 1; Gia-Shing Shieh 2; Chrong-Reen Wang 3; Ai-Li Shiau 4 1National

Cheng Kung University Medical College, Biochemistry and Molecular Biology, 1, Dashiue Road, Tainan, Taiwan; 2Tainan Hospital, Department of Health, Urology, Tainan, Taiwan; 3National Cheng Kung University Medical College, Internal Medicine, 1, Dashiue Road, Tainan, Taiwan; 4National Cheng Kung University Medical College, Microbiology and Immunology, 1, Dashiue Road, Tainan, Taiwan Background: Synovial fibroblasts (SFs) that mediate bone erosion and joint destruction play a pivotal role in the pathogenesis of rheumatoid arthritis ( RA) and thus represent an ideal therapeutic target. Giving that the phenotype of rheumatoid synovium is similar in many respects to that of an aggressive tumor, including p53 mutation and high telomerase activity, here we examined the feasibility of using a telomerase-dependent oncolytic adenovirus as a therapeutic strategy in a rat model of collagen-induced arthritis (CIA). Methods: The p53 status and telomerase expression of SFs from patients with RA and arthritic rats were examined with immunohistochemistry, immunoblotting, or luciferase reporter assays. Ad.GS1, an E1B 55 kD-deleted adenovirus driven by the human telomerase reverse transcriptase (hTERT) promoter, was injected intraarticularly to rats with CIA and their treatment responses were monitored. Expressions of cytokines and enzymes associated with RA were also determined. Results: SFs from patients with RA and arthritic rats exhibited p53 mutation and telomerase activity compared with SFs from patients with osteoarthritis or from normal rats. Ad.GS1 replicated in and induced cytolysis of SFs from human and rat samples but spared normal fibroblasts. Furthermore, Ad.GS1 treatment significantly reduced ankle circumference, articular index scores, radiographic scores, and histologic scores, as well as decreased production of interleukin-1, matrix metalloproteinase-9, and prolyl 4-hydroxylase in rats with CIA compared with Ad.LacZ or PBS treatment that served as controls. However, the levels of macrophages infiltrating into the ankle joints and tumor necrosis factor- were unaffected. Productive replication of Ad.GS1 was also detected in the synovial tissue in the treated rats.

1162 Conclusion: These results are the first to demonstrate the therapeutic effects of telomerase-dependent oncolytic adenovirus on amelioration of arthritic symptoms in rats with CIA. Our findings also implicate that oncolytic adenoviruses may be further explored as a therapeutic agent for RA. E-mail: [email protected] P 171 Insertion of E2F-responsive sites in E1a promoter to improve the efficacy to toxicity ratio of oncolytic adenoviruses Juan J Rojas ; Manel Cascallo ; Sonia Guedan ; Alena Gros ; Francisca Alcayaga ; Ramon Alemany IDIBELL-Institut Catala d’Oncologia (ICO), Translational Research Laboratory, Av Gran Via s/n km 2,7, L’Hospitalet de Llobregat. 08907-Barcelona, Spain E2F-responsive sites play a double role in promoters: restrict transcription when E2F is forming a complex with functional pRb, as in normal cells, and promote transcription when E2F is released due to a deregulation of pRb pathway, as in cancer cells. We took advantage of this characteristic to modify the promoter controlling E1a transcription by the insertion of E2F-responsive sites to construct more selective and potent oncolytic adenoviruses. ICOVIR-7 is a new oncolytic adenovirus that controls the expression of Kozak-E1a24 gene under a modified E2F-1 promoter (E2F-responsive promoter) insulated with the myotonic dystrophy locus insulator DM-1. However, the different genetic elements present in ICOVIR-7 result in a high genome size barely able to harbor a transgene if we want to improve the spreading or the cytotoxicity. To construct a more compacted backbone, Ad41524RGD was created by the insertion of E2F-responsive sites in the endogenous E1a promoter. Selectivity was tested in a liver slices approach and after systemic administration of viruses in immunocompetent mice. Replication in tumor cells and efficacy in subcutaneous xenograft tumors after systemic administration were assessed as oncolytic potency tests. The insertion of the new E2F-responsive sites resulted in improved oncolysis and restriction of the replication in normal cells, claiming these new viruses as a promising tool for treating disseminated neoplasias. E-mail: [email protected] P 172 Intravenously administered alphavirus vector VA7 eradicates orthotopic human glioma xenografts in nude mice Jari Heikkila 1; Markus Vaha-Koskela 2; John C. Bell 2; Ari Hinkkanen 3 1Abo

Akademi University, Biochemistry & Pharmacy, Turku, Finland; 2University of Ottawa, Ottawa Health Research Institute, Ottawa, Canada; 3A.I. Virtanen Institute, Biotechnology and Molecular Medicine, Kuopio, Finland Background: Malignant gliomas are the most common primary brain tumours in adults and unfortunately, no significant progress has been achieved during the past decades in the treatment of these cancers. We have previously shown that attenuated replication competent alphavirus vector VA7 exhibits oncolytic activity against human melanoma xenografts in immunocompromised mice. Since VA7 is neurotropic and is able to deliver therapeutic genes into the CNS of mice upon systemic administration, we sought to investi-

ESGCT 2008 POSTER PRESENTATIONS gate whether VA7 would be effective against human glioma xenografts when administered intravenously in an animal model of human glioma. Methods: Firefly luciferase expressing human glioma cells U87Fluc were implanted intracranially in Balb/c nude mice. Tumour growth was monitored by injecting the mice with firefly luciferin and the light produced was detected by the IVIS Imaging System. In order to study the biodistribution of VA7 we cloned Renilla luciferase gene into VA7 and the VA7Rluc vector was injected intravenously into naïve and tumour bearing mice. VA7Rluc replication was monitored by injecting the animals with coelenterazine, a substrate of Renilla luciferase, and the light produced was detected by the IVIS Imaging System. Results: A single intravenous injection of VA7-GFP caused in 6 days a complete quenching of intracranial firefly luminescence in U87Fluc tumour-bearing mice. No regrowth of tumours was observed during a 100-day follow-up and post mortem histology revealed no signs of microscopic tumours. We next injected U87Fluc tumour-bearing mice with VA7Rluc and studied the tumour selectivity of the virus. Intracranial VA7Rluc luminescence was observed in all tumour bearing mice indicating VA7Rluc replication in the tumour. In some animals weak, transient peripheral Renilla luminescence was observed. No Renilla luminescence could be detected in naïve, non-tumour bearing mice. Conclusion: VA7 rapidly and effectively eradicates intracranial human glioma xenogratfs and does not show significant non-specific peripheral replication or adverse effects. E-mail: [email protected] P 173 Effect of decorin on overcoming the extracellular matrix barrier for oncolytic virotherapy Joo-Hang Kim 1; Il-Kyu Choi 1; Young-Sook Lee 1; Ji Young Yoo 1; A-Rum Yoon 1; Hye Jin Choi 2; Chae-Ok Yun 1 1Yonsei University College of Medicine, Institute for Cancer Research, Seoul, Republic of Korea; 2Yonsei University College of Medicine, Internal Medicine, Seoul, Republic of Korea

The pressing challenge for contemporary gene therapy is to deliver enough therapeutic genes to enough cancer cells in vivo. With the aim of improving viral distribution and tumor penetration, we explored the use of decorin to enhance viral spreading and tumor tissue penetration. We generated decorin-expressing replication-incompetent (dl-LacZDCNG, dl-LacZ-DCNQ, and dl-LacZ-DCNK) and -competent (Ad-E1B-DCNG, Ad-E1B-DCNQ, and Ad-E1BDCNK) adenoviruses. Point mutants of decorin gene (DCNG), DCNK and DCNQ, has a negative and moderate binding affinity to type I collagen fibril, respectively. In both tumor spheroids and established solid tumors in vivo, tissue penetration potency of dl-LacZ-DCNG is greatly enhanced than those of dl-LacZ, dl-LacZ-DCNQ, and dl-LacZ-DCNK, and this enhanced tissue penetration effect derived from decorin-expressing adenovirus is dependent on the binding affinity of decorin to collagen fibril. Expression of DCNG enhanced viral spread of replicating adenovirus, leading to improved tumor reduction and survival benefit. Moreover, the tumoricidal effects of Ad-E1B-DCNQ and Ad-E1B-DCNK was decreased as the binding affinity to collagen was decreased, demonstrating that the increase of cancer cell killing effect is driven by decorin’s action on extracellular matrix. Furthermore, Ad-E1B-DCNG substantially decreased ex-

ESGCT 2008 POSTER PRESENTATIONS tracellular matrix components within the tumor tissue. Finally, intratumoral injection of Ad-E1B-DCNG in primary tumor site greatly reduced the formation of pulmonary metastases of B16BL6 melanoma cells in mice. Taken together, these data demonstrate the utility of decorin as a dispersion agent and suggest its utility and potential in improving the efficacy of replicating adenovirus-mediated cancer gene therapy. E-mail: [email protected] P 174 The selective oncolytic effect of Newcastle Disease Virus (NDV-HUJ) relates to a higher replication of the virus and to unique pathways in the tumor cell Barak Yaacov 1; Itay Lazar 2; Michal Lotem 2; Zichria Zakay-Rones 1; Riki Perlman 2; Dina Ben-Yehuda 2; Amos Panet 1 1The

Hebrew University-Hadassah Medical School, Virology, Jerusalem, Israel; 2Hadassah Medical Center-Hebrew University, Hematology, Jerusalem, Israel Newcastle Disease Virus (NDV) has been shown in preclinical and clinical trials to selectively induce the killing, oncolysis, of tumor cells. Yet, the mechanism of viral induced oncolysis is not clear. We investigated the mechanism of oncolysis by an attenuated isolate of NDV (HUJ) in two human cancers, lung carcinoma and melanomas. We found in both cell systems that viral replication, i.e RNA transcription, and protein translation are higher in the tumor cells as compared to the non-tumoregenic cells and that the virus induces the intrinsic apoptotic cascade selectively in the tumor cells. Furthermore, we demonstrated that primary cells derived from patients with advanced melanoma are much more sensitive to NDV-HUJ induced apoptosis as compared to cells derived from early melanomas. The selective killing of advance melanoma cells was found related to over-expression of the antiapoptotic protein Livin in the advanced melanomas but not in the early melanomas. NDV-HUJ infection specifically induces a limited proteolysis/ truncation of the Livin protein to convert into a potent pro-apoptotic protein termed t-Livin, that in turn accelerates apoptosis. These original observations demonstrate the ability of an oncolytic virus to exploit the bi-functional role of Livin as an apoptosis regulator in the treatment of tumors. The data presented here indicate the potential of the oncolytic NDV-HUJ for the treatment of advance melanoma that is otherwise extremely resistant to all known chemotherapy treatments. E-mail: [email protected] P 175 Tuning of oncolytic adenovirus magnetic complexes: Tumor-killing effect on CAR-deficient multidrugresistant cancer cells Nittaya Tresilwised 1; Olga Mykhaylyk 2; Martina Anton 2; Regina Holzmueller 2; Pimolpan Pithayanukul 1; Per Sonne Holm 2; Christian Plank 2 1Mahidol University, Faculty of Pharmacy, Department of Pharmacy, Bangkok, Thailand; 2Klinikum Rechts der Isar, Institute of Experimental Oncology, Munich, Germany

Multidrug resistance is a major obstacle to the success of conventional cancer therapies. Oncolytic adenoviruses, a new class of biological anti-cancer agents, are developed to

1163 lyse tumour cells upon virus replication, spread to neighbouring tumour cells and continue the lytic cycle. Recently, it was discovered that nuclear localization of the transcription factor Y-box binding protein (YB-1) plays an essential role in tumour-selective adenoviral replication leading to specific oncolysis. In many tumour cell types, adenoviral infection is limited by lacking expression of the coxsackie and adenovirus receptor (CAR). We have previously shown that this limitation can be overcome by magnetofection. In the present work, we studied association of the YB-1-dependent oncolytic adenovirus Ad520 with a set of polycation-coated iron oxide magnetic nanoparticles and characterized magnetic virus complexes in terms of their size, magnetic moment and compositions. We established relationships between virus dose, magnetic particle-to-virus particle ratio and oncolytic effect in cultured daunorubicin-resistant human pancreatic cancer cells, which are CAR deficient. Internalization of virus and magnetic nanoparticles into cells was quantitatively characterized. Magnetic virus complexes were significantly more efficient to internalize into the cells as compared to the native virus. No inhibition of the virus activity was due to the association with magnetic nanoparticles. We found that optimal compositions of the complexes under magnetofection conditions culminated in a great increase in the oncolytic potency and kinetics of oncolysis in contrast to native virus. Using non-replicating adenovirus as a control, we demonstrated that the oncolytic effect is entirely due to the increased virus replication and not to unspecific toxic effects of the magnetic virus complexes. Ongoing studies will demonstrate whether the observed magnetic boosting of oncolytic adenovirus can be translated to in vivo therapy in a CAR-deficient multidrug resistant mouse tumour model. E-mail: [email protected] P 176 Low dose antitumor efficacy of a targeted and armed oncolytic vaccinia virus for renal cell cancer Kilian Guse ; Marta Sloniecka ; Iulia Diaconu ; Vincenzo Cerullo ; Akseli Hemminki University of Helsinki, Cancer Gene Therapy Group, Helsinki, Finland Background: Vaccinia virus has strong oncolytic potency due to its high transduction efficiency and rapid replication cycle making it a promising anticancer agent. Although vaccinia virus has been injected in millions of humans during the smallpox eradication program, safety concerns remain especially when administered systemically in high doses for cancer treatment. Thus, transcomplementational targeting strategies to restrict replication to tumor cells have been developed. To enhance antitumor effect, vaccinia virus can be armed with therapeutic transgenes such as soluble VEGF receptor 1 (sFlt) as an antiangiogenic approach which might be particularly useful for kidney cancer. We hypothesized that a transcomplementationally targeted oncolytic vaccinia virus armed with sFlt would show improved anticancer efficacy in s.c. mouse models. Methods: vvdd-sFlt, a western reserve vaccinia virus featuring deletion in thymidine kinase and vaccinia growth factor genes for cancer cell targeting was constructed. Efficacy of this virus was compared to vvdd-luc, an isogenic nonarmed control virus, in vitro and in vivo in s.c. models using different doses and routes of injection. Cytokine profile of vvdd-sFlt injected mice was assessed by FACSarray.

1164 Results: In vitro, vvdd-sFlt showed efficient transduction and strong oncolytic effect on renal cancer cells. In immunodeficient in vivo kidney cancer models, sFlt expression in the blood was confirmed by elisa and antiangiogenic effect was seen. vvdd-sFlt and vvdd-luc were able to significantly reduce tumor size after i.t. injection of high and medium doses. However, only vvdd-sFlt was able to reduce tumor size after low dose i.t. injection. High dose i.v. injection of vvdd-sFlt and vvdd-luc resulted in tumor size reduction but only vvdd-sFlt was effective in medium dose i.v. model. Lethal toxicity after high dose virus injection was seen in some mice, whereas medium and low doses were well tolerated. Cytokine profiles revealed that a marked reduction of inflammatory markers can be achieved by lowering virus dose. Studies with immunocompetent, syngeneic, kidney cancer mouse models are underway. Conclusion: vvdd-sFlt is an efficient oncolytic virus which has antitumor activity even at low non-toxic doses. E-mail: [email protected] P 177 Matrix metalloprotease-activation prevents destruction of healthy human liver tissue by oncolytic measles virus Michael Muehlebach 1; Thomas Schaser 1; Martina Zimmermann 2; Sorin Armeanu 2; Michael Bitzer 2; Ulrich Lauer 2; Roberto Cattaneo 3; Christian Buchholz 1; Klaus Cichutek 1 1Paul-Ehrlich-Institut, Div. of Medical Biotechnology, Langen, Germany; 2Medizinische Universitätsklinik, Innere Medizin, Tübingen, Germany; 3Mayo Clinic, Dep. of Molecular Medicine, Rochester MN, United States

Oncolytic viruses conditionally replicating in cancer cells are an attractive novel class of agents for the therapy of solid tumours. Making the cell entry process of measles virus (MV) dependent on tumour-associated matrix metalloproteases (MMPs) restricts virus propagation to tumour cells and improves the virus safety in xenograft tumour models (1). With a view to targeting solid liver tumours, protease expression of patient material was investigated. In liver tumour sections, high levels of MMP2 and overexpression of MMP8 and TIMP-2 were detected by zymography or protein arrays, respectively. Three MMP cleavage sites previously selected from retroviral protease substrate libraries on cell lines overexpressing MMP-2 and TIMP-2 were inserted into the MV fusion protein F, which requires activation by protease cleavage. MMP-dependent protease activation was analysed and genes for MMP-activatable F proteins were inserted into GFP encoding measles virus (MV) genomes. For the MVMMPA1 virus (1), activation could be attributed to MT1MMP. As expected, the MV-MMPA1 virus was unable to spread through primary human hepatocytes, while unmodified MV-GFP readily propagated on these cells, as detected by GFP- and nucleocapsid protein expression. Both viruses were further characterized on cultivated slices prepared from tumour bearing liver of hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) patients. For patients S002 (CRC) and S003 (HCC), numerous GFP-expressing foci were detected in the tumour slice, while in S002 unaffected liver was GFP-negative, being in line with MMP2 processing indicating active MT1-MMP. In non-cancerous liver tissue of patients S026 (HCC) and S028 (CRC) spreading of MVMMPA1 was strongly restricted, whereas large areas of massive syncytia were caused by the MV-GFP virus. The data demonstrate that MMP activation can efficiently

ESGCT 2008 POSTER PRESENTATIONS restrict virus spreading to MMP-positive tumour tissue and support the application of MMP activatable oncolytic viruses for liver cancer therapy. (1) Springfeld et al., Cancer Research 2006, p. 7694-7700. E-mail: [email protected] P 178 Late expression of a fusogenic glycoprotein enhances the cytotoxicity of an oncolytic adenovirus deleted in E4orf1/2/3 Sonia Guedan 1; Alena Gros 1; Manel Cascallo 1; Richard Vile 2; Elena Mercade 3; Ramon Alemany 1 1Institut Català d’Oncologia, Laboratori de Recerca Translacional, L’Hospitalet de Llobregat, Spain; 2Mayo Clinic, Molecular Medicine Program, Rochester, United States; 3University of Barcelona, Department of Microbiology, Barcelona, Spain

Background: We have previously described ICOVIR5 (Ad-DM-E2F-K-d24RGD), a potent and selective oncolytic adenovirus that expresses a mutated E1a under the control of an insulated form of the E2F promoter. Here, we aimed to develop a version of this virus with increased spreading capacity through the expression of the fusogenic glycoprotein of the gibbon ape leukemia virus (GALV) during the late stage of virus replication. Methods: ICOVIR9, the ICOVIR5 derivative armed virus, was constructed by expressing GALV under the control of the major late promoter. In order to accommodate the insertion of GALV cDNA without affecting encapsidation, E4orf1, E4orf2 and E4orf3 genes were deleted from ICOVIR9. An Adwtorf123del virus lacking the E4orf123 genes was also constructed in order to assess the effect of this deletion. Cytopathic effect, cell killing, and virus production was assessed in a wide range of tumor cells infected with Adwtorf123del and ICOVIR9 compared with their parenteral viruses, Adwt and ICOVIR5. Finally, the in vivo efficacy of the fusogenic oncolytic adenovirus compared with ICOVIR5 was compared using a subcutaneous tumor xenograft model in nude mice. Results: Adwtorf123del showed a slight decrease in viral replication and viral cytotoxicity in all tumor cells. In agreement with these results, ICOVIR9 showed lower viral production yields per infected cell compared to ICOVIR5. Despite having such a lower burst size, ICOVIR9 was more cytotoxic than ICOVIR5 and induced extensive syncytia formation. In vivo administration of ICOVIR9 into nude mice bearing tumor xenografts led to the improved control of tumor growth. Conclusion: Late expression of GALV glycoprotein enhances the efficacy of oncolytic adenovirus. However, shorter insulators/promoters or additional deletions of viral DNA other than E4 orfs1/2/3 need to be investigated to provide genomic capacity compatible with efficient virus production. E-mail: [email protected] P 179 Type 5 adenoviruses bearing the type 35-derived fiberknob region enhanced the infectivity and increased the anti-tumor effects to CAR-low expressing tumors Masatoshi Tagawa 1; Kiyoko Kawamura 1; Guangyu Ma 1; Quanhai Li 1; Yuki Takei 1; Nobuo Suzuki 2; Naoto Yamaguchi 3; Yuji Tada 4; Kenzo Hiroshima 5; Hideaki Shimada 6

ESGCT 2008 POSTER PRESENTATIONS 1Chiba

Cancer Center Research Institute, Division of Pathology, Chiba, Japan; 2Graduate School of Medicine, Chiba University, Department of Environmental Biochemistry, Chiba, Japan; 3Graduate Sch of Pharm Sciences, Chiba University, Department of Molecular Cell Biology, Chiba, Japan; 4Graduate School of Medicine, Chiba University, Department of Respirology, Chiba, Japan; 5Graduate School of Medicine, Chiba University, Department of Diagnostic Pathology, Chiba, Japan; 6Chiba Cancer Center Research Institute, Division of Gastroenterological Surgery, Chiba, Japan Gene transfer with adenoviruses type 5 (Ad5) vectors is influenced by expression levels of Ad receptors on target cells. Ad5 use the coxsackievirus and adenovirus receptor (CAR) as the primary receptor, which interacts with Ad fibers-knob regions. The expression level of CAR molecules is often downregulated in human tumors, which consequently hampers Ad5-mediated gene transfer. Ad type 11 (Ad11) and type 35 (Ad35), belonging to a subtype B group, use CD46 molecules as their cellular receptors and the expression level is well maintained in human tumors. Chimeric Ad5 whose fiber structure was substituted with that of the type 11 or 35 (Ad5/F11 or Ad5/F35) could infect human cells in a different manner from Ad5 and showed greater transduction capability in particular to tumors. We constructed the chimeric Ad5 bearing the green fluorescence protein gene and found with the Ad that Ad5/F35 and Ad5/F11 infected human tumors better than Ad5. Infection of Ad5 suppressed subsequent gene transfer with Ad5 but not with Ad5/F11 or Ad5/F35, and infection of Ad5/F35 down-regulated following transduction with Ad5/F35 and Ad5/F11 and to lesser extents with Ad5. The precedent Ad infection decreased the receptor expression level and interfered with subsequent gene transfer mediated by the same Ad vector. These data collectively suggest that combinatory use of Ad 5 and the chimeric Ad enables dual gene transfer into target cells. We also prepared chimeric Ad5 in which the E1A expression was regulated by an exogenous promoter that was active in human tumors. The fiber-modified replication-competent Ad produced similar cytotoxic activities as prototype Ad5 to mesothelioma with CAR-high expression and achieved greater anti-tumor effects than the Ad5 to mesothelioma with CAR-low expression, suggesting a possible use of chimeric Ad for cancer therapy in particular to CAR-low tumors. E-mail: [email protected] P 180 Prostate cancer targeting by replication-selective adenoviral mutants: combination treatments with phytochemical drugs Virginie Adam ; Nicolas Lemoine ; Gunnel Hallden Barts and The London School of Medicine, Cancer Research UK, Centre for Molecular Oncology, London, United Kingdom Background: Replication-selective adenoviruses are a promising new type of therapy for androgen-independent prostate cancer (PCa). Numerous studies demonstrated that the anti-tumour potency of adenoviruses was greatly enhanced by combination treatments with conventional anticancer treatments including chemotherapy and radiotherapy. Here we describe how the non-toxic phytochemicals equol and resveratrol act synergistically with adenovirus type 5 (Ad5) to kill PCa cells. Methods: The effects of equol and resveratrol with and without Ad5 on cell survival were analysed by MTS assay in the AR-positive 22Rv-1 and LNCaP cells, and the AR-neg-

1165 ative PC-3 and DU145 cells, to determine EC50 doses and combination indexes. Flow cytometry was used to analyse the infectability of cells using a non-replicating Ad5-GFP and the cellular expression levels of adenoviral receptors. The effects of phytochemicals with and without Ad5 on cell cycle progression and cell death was also investigated by flow cytometry, immunoblotting and confocal microscopy. Results: Synergistic enhancement of cell death was detected in cells treated with combinations of equol (10-100M) or resveratrol (5-15M) and Ad5 (0.1-160 particles per cell). Both phytochemicals also significantly increased the infectability of PCa cell-lines. In DU145 the percentage cells infected increased from 38% (100ppc) to 42% (100M equol) and 55% (10M resveratrol). The enhanced infectability was paralleled by increased expression of Ad5 receptors on the cell surface. In DU145 cells, the surface expression level of V3 increased from 44% to 58% in equol-treated cells (100M) and to 67% in resveratrol-treated cells (10M). Although apoptosis is induced by the phytochemicals alone, the synergistic mechanism of cell death does not solely rely on this pathway. Conclusion: Equol and resveratrol can synergistically induce cell death with Ad5 and cancer-selective mutants in AR and AR-PCa cell-lines. The phytochemicals enhance infectability but do not affect viral replication. The cell death mechanism is currently under investigation. E-mail: [email protected] P 181 The development of replication-competent adenovirus targeting bladder and prostate cancer: Adenoviral early E1a and E4 protein expression are regulated by cancer specific modulated mode Jae Kook Nam 1; Mi Hyang Lee 2; Moonjae Cho 3; Sang Jin Lee 4 1Cheju

National University School of Medicine, Pediatrics, Jeju, Republic of Korea; 2National Cancer center, Genitourinary Cancer Branch, Translational and Clinical Research, Goyang, Republic of Korea; 3Cheju National University School of Medicine, Biochemistry, Jeju, Republic of Korea; 4Genitourinary Cancer Branch, Translational and Clinical Research, Goyang, Republic of Korea Adenovirus-based gene therapy is an engaging approach for the treatment of inveterate neoplastic diseases. Specially, conditionally replicating adenoviruses have studied extensively because of its usability and have been widely applied in cancer gene therapy. It is obvious that these therapeutic techniques are very useful, but a lot of effort and time are required for the correct establishment of these techniques. The aim of this study was development of oncolytic adenovirus capable of replication selectively in cancer cells. We have developed a recombinant adenovirus (Ad5/35E4psurvivinE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer, rat survivin promoter and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of survivin is abundantly expression in most of cancer cells but is scarcely any expression in non-neoplastic cells. Ad5/35E4psurvivinE1a was replaced to Ad35-type fiber, so the efficiency of viral infectivity was greatly improved in bladder cancer cell lines (253J, J82, and T24). Also, Ad5/ 35E4psurvivinE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in bladder cancer cell lines and prostate cancer cell lines (CWR22rv,

1166 LNCaP, PC3, and Du145), but it hardly affected to L132, transformed lung epithelial cell line. Also, we tried to investigate protein expression pattern. The E1a and E4 of Ad5/35E4psurvivinE1a were overexpressed in cancer cell lines but were rarely expressed in L132. It was corresponded with results of the cell level. Through these results, we have confirmed that Ad5/35E4psurvivinE1a specifically works to cancer cell. In summary, adenoviral replication could be controlled by bidirectional regulation of E1a and E4 gene with a single enhancer, rat survivin promoter. Also, the recombinant adenovirus (Ad5/35E4psurvivinE1a), which is made in previously described manner, works to not tissue-specific but tumor-specific. And therefore Ad5/35E4psurvivinE1a would be useful in a treatment of various tumors, which are irrelevant to origin. E-mail: [email protected] P 182 Development of an EIAV based lentiviral vector producer cell line Hannah Stewart ; Kyriacos Mitrophanous ; Pippa Radcliffe Oxford BioMedica (UK) Limited, Virology, Oxford, United Kingdom The large-scale production of Equine Infectious Anaemia virus (EIAV)-based lentiviral vectors would benefit greatly from the development of producer cell lines as this would enable the collection of large quantities of vector over extended periods. Such cell lines would contain three vector components (Gag/Pol, VSV-G envelope and genome constructs) stably integrated into the cellular chromosomes. A complicating factor is that the VSV-G envelope protein is cytotoxic and therefore its expression must be regulated. It is also desirable to regulate Gag/Pol expression to minimise metabolic burden on the cell. The Tet Repressor (TetR) system has been selected to regulate expression of VSV-G and Gag/Pol. This requires the introduction of a fourth construct encoding TetR, into the cell line. ProSavin® is Oxford BioMedica’s EIAV-based therapy for Parkinson’s disease, and here we describe the development of an inducible ProSavin HEK293T based producer cell line. This cell line demonstrated excellent TetR controlled regulation of the packaging components. On induction, vector was produced and titres were comparable to the transient system. The cell line proved to be stable (sixty days in culture) and capable of producing high titre vector. E-mail: [email protected] P 183 Nonclinical supporting studies for a first in man clinical trial using direct administration of a lentiviral vector (ProSavin®) for the treatment of Parkinson’s disease James Miskin 1; Debbie Day 2; Georgina Ferrige 1; Julie Loader 1; Diana Angell-Manning 1; Margaret Esapa 1; Kyriacos Mitrophanous 1 1Oxford

BioMedica plc, Virology, Oxford, United Kingdom; BioMedica plc, Clinical Development, Oxford, United Kingdom 2Oxford

Background: Lentiviral vectors hold great promise for the treatment of human diseases, as they transduce post-mitotic

ESGCT 2008 POSTER PRESENTATIONS cells leading to long-term expression. A phase I/II first in man (FIM) clinical trial for the treatment of Parkinson’s disease is ongoing3 using direct intra-striatal administration of ProSavin, an EIAV-based lentiviral vector that converts cells into dopamine factories. ProSavin corrected a variety of motor deficiencies in the non-human primate (NHP) MPTP model of Parkinson’s disease for up to 28 months. Additional nonclinical studies performed prior to gaining regulatory approval for the ProSavin FIM study are briefly summarised below. Results:Vector shedding and dissemination was investigated in the rat at 1 hour, 24 hours and seven days (qPCR); approximately 0.5% of the administered vector was detected in the CSF at one hour, which reduced 800-fold over 24 hours. On average 0.1% of the administered vector was detected in scalp wound swab samples after surgery; this reduced ten-fold using disinfectant and reduced further over time. Very low levels of vector were detected in plasma and urine at 1 and 24 hours, and were not detected after seven days. Biodistribution was investigated in the rat at 3 days, 30 days and 6 months (qPCR). Tissues analysed were brain (left and right striatum), kidneys, lungs, lymph nodes, blood, liver, spleen, heart, spinal cord and gonads. ProSavin was found mainly in the brain. Very little ProSavin crossed the blood-brain barrier and, then was found in very low quantities when compared with the administration site. Toxicology was assessed in rat and NHP; in-life monitoring and toxicological endpoints revealed no ProSavin-related toxicities or adverse effects. Antibodies to VSV-G were detected in a subset of animals, but not to other structural components or the transgenes. Real-time PCR analysis revealed insignificant ProSavin-derived circulating vector RNA and DNA; vector RNA was not detected in the CSF. 3Prof. Stéphane Palfi, Principle Investigator, Henri Mondor Hospital, France (EudraCT Number: 2007-001109-26) E-mail: [email protected] P 184 Non-invasive in vivo imaging of somatostatin receptor 2 gene transfer with positron emission tomography Gabriella Cotugno 1; Armida Faella 1; Michela Aurilio 2; Valentina Rinaldi 2; Maurizio Di Tommaso 1; Luigi Aloj 2; Alberto Auricchio 1 1Telethon Institute of Genetics and Medicine, TIGEM, Naples, Italy; 2I.N.T. Fondazione “G. Pascale”, Department of Nuclear Medicine, Naples, Italy

Non invasive imaging of gene transfer is useful for follow up of gene therapy protocols in animal models and then in patients. To this aim, reporter transgenes with low endogenous expression levels allowing specific in vivo non-invasive detection of gene expression are desirable. The human somatostatin receptor 2 (SSTR2) has very low expression levels in a variety of tissues including muscle, liver or lung and has been used as reporter for in vivo imaging after tumordirected viral-mediated gene delivery. Several somatostatin analogues labelled with radioactive isotopes have been used for in vivo detection of SSTR2 gene expression with various imaging techniques, including Positron Emission Tomography (PET). Vectors derived from the non-human pathogenic adenoassociated virus (AAV) are ideal for long-term in vivo gene transfer to animal models and to humans. We have tested the possibility to non-invasively and quantitatively monitor

ESGCT 2008 POSTER PRESENTATIONS gene transfer by PET imaging of the human SSTR2 transgene following AAV-mediated gene transfer to different tissues and using 68gallium-DOTA-Tyr(3)-Thr(8)-octreotate (Ga-68 labelled DOTATATE) as highly specific SSTR2 ligand. We initially showed hSSTR2 expression in transfected cells by immunofluorescence; we then demonstrated that recombinant SSTR2 bound in vitro with high specificity the SSTR2 ligand In-111-Octreoscan. We then administered to muscle or liver of C57/BL6 mice AAV vectors encoding SSTR2 under the control of either a ubiquitous or of the liver-specific promoter TBG (tyroxine-binding globulin) respectively. A month after vector administration following systemic injection of Ga-68-DOTATATE, specific signal corresponding to SSTR2 expression in liver and muscle transduced with AAV was evident by PET imaging. Quantitative measurement of Standardized Uptake Values (SUV) showed increased SUV in AAV-transduced muscles and livers compared to controls (4 and 6 fold increase in muscle and liver respectively). Experiments aimed at assessing the AAV vector dose-PET imaging response are ongoing as it is imaging AAV-mediated lung SSTR2 expression by PET. In conclusion the AAV-SSTR2/ Ga-68-DOTATATE system represents a promising tool for non-invasive long-term monitoring of gene transfer by PET. E-mail: [email protected] P 185 The role of free polyethylene imine (PEI) in gene delivery by DNA-PEI complexes Martina Hanzlikova 1; Emilia Galli 2; Arto Urtti 3; Atso Raasmaja 1; Marika Ruponen 4; Marjo Yliperttula 2 1University

of Helsinki, Division of Pharmagology and Toxicology, Faculty of Pharmacy, Helsinki, Finland; 2University of Helsinki, Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, Helsinki, Finland; 3University of Helsinki, Centre for Drug Research, Faculty of Pharmacy, Helsinki, Finland; 4University of Kuopio, Department of Pharmaceutical Technology and Biopha, Faculty of Pharmacy, Kuopio, Finland

Background: Polyethylene imine (PEI) is a cationic polymer which has been widely used in non-viral gene delivery. Cationic PEI forms spontaneously complexes with anionic DNA. The best gene transfer is seen at high N/P ratios with excess of PEI. However, the exact role of the excess PEI of the DNA complexes is not clearly understood. Anionic glycosaminoglycans (GAGs) expressed on the cell surfaces and extracellular space interact with cationic gene delivery complexes. The purpose of this study was to examine the correlation of the free carrier and the role of GAG content of the cells in the delivery process and to further understand in more detail the role of the free carrier in the gene delivery process. Methods: Free PEI was removed from the PEI complexes by size exclusion chromatography1. The transfection efficiency and toxicity assays with purified (without free PEI) and non-purified complexes (with free PEI) were performed using smooth muscle cells (SMC), Chinese hamster ovary (CHO) cells, and GAG-deficient mutant cells (CHO-618) derived from the CHO cell line. Moreover, the intracellular elimination of pDNA after transfections with purified and non-purified complexes was evaluated by qRT-PCR method. Results: The transfection efficiency of non-purified PEI complexes was significantly higher than that of purified PEI complexes in SMC cells and CHO cells with GAGs. However, in CHO-618 cells without GAGs the transfection effi-

1167 ciency of purified and non-purified PEI complexes was at the same level. Interestingly, addition of free PEI to purified PEI complexes increased transfection efficiency to the same level with non-purified PEI complexes in GAG containing cells. Toxicity assays showed that free carrier increased the toxicity in the cells. Conclusion: Free carrier improves transfection efficiency of PEI complexes. This may be mechanistically related to the GAG content of the cell surface. 1Boeckle at al. (2006) J Gene Med; 6, 1102-1111. E-mail: [email protected] P 186 Topical delivery of antisense oligonucleotide using chitosan nanoparticles Suna Ozbas-Turan ; Julide Akbuga ; Ali Demir Sezer Marmara University, Faculty of Pharmacy, Pharmaceutical Biotechnology, Istanbul, Turkey Despite the development of various delivery carriers for antisense oligonucleotides (AsODNs), few successful cases have been reported of skin application of AsODN. We have previously developed and investigated the in vitro effect of AsODN-loaded chitosan nanoparticles. The purpose of this study was to investigate in vivo antisense effect of AsODN-loaded chitosan nanoparticles after topical application. 15-nucleotide phosphorothioate oligonucleotide (MWG-Biotech Germany) designed to target gal gene and chitosan (400 kD, Fluka) and tripolyphosphate were used for nanoparticle preparation. AsODN encapsulated TPP-chitosan nanoparticles were topically applied to Spraque Dawley rats (8 weeks old adult and 4 weeks old baby). One, 3 and 6 days of post-transfection animals were sacrified and skin samples were taken for measurement of -gal expression and histological control. gal expression was spectrophotometrically measured in the samples with ONPG. Total protein concentration was assayed according to Bradford’s method. Untreated group of animals was used as a control. After topical application of AsODN nanoparticles in different doses (15, 30, 45, 60 and 90 g) -gal expression reduced significantly (p  0.001). Highest inhibition was observed after 6 days of transfection of nanoparticles. Free AsODN exhibited 34% of -gal inhibition in the first day then the reducing effect decreased. However nanoparticles exhibited an increasing antisense effect. -gal expression was inhibited in approximately 8285%. A dose-dependent antisense effect was not found with AsODN nanoparticles and no difference was also observed between the values of adult and baby rats. Thus, chitosan-TPP nanoparticles are useful carrier for delivery of antisense oligonucleotides into skin cells of rats and may be used for topical application on human skin. Acknowledgements: This study was supported by Commission of Marmara University Scientific Research Project (BAPKO, SAG-BGS-120707-0143). E-mail: [email protected] P 187 Cellular and molecular characterization of spontaneous replication competent adenoviruses in the production of helper-dependent adenoviral vectors Idalia Dávila-González ; Angélica Ortega-García ; Angélica Meneses-Acosta

1168 Universidad Autónoma del Estado de Morelos, Faculty of Pharmacy, Cuernavaca, Mexico Background: Adenoviral vectors (AdV) have drawn great attention in gene therapy given their advantages related to the high titers obtained and their null association with any neoplastic disease. Different molecular designs were developed thus resulting in several generations of AdV. The presence of Spontaneous Replication Competent Adenovirus (SRCAs) is a limitation due to the biosafety and has been characterized by infection of AdV in permissible cells and by PCR. Though the SRCA’s could have any fragment of the viral genome, the most pertinent SRCAs are adenoviral particles that acquire the E1 region from the host cell by homologous recombination. To date, efforts have been focused to identify the SRCAs in first and second generation AdV but third generation adenoviruses have not had enough attention. Therefore, the objective of this work was to identify and to quantify the SRCAs generated during the propagation of the helper virus of the FLPe/frt system in HEK293 cells for the production of helper-dependent adenoviral vectors. Methods: Helper virus amplification up to eight passages by means of consecutive infections by the virus in HEK293. Identification and quantification of Total Viral Particles (TVP) and Infective Viral Particles (IVP) by HPLC and End Point Dilution techniques, respectively. Identification of SRCAs by means of bioassay in three stages using permissible HeLa and A549 cells. PCR using 2 sets of primers for the E1 region. Electron microscopy developed by standard methods. Results: Viral titers obtained from the eight amplifications of helper virus oscillated between the reported values. Each helper virus passage was used to infect HeLa and A549 cells at MOI of 100. In both cell lines, generation of lytic plates were observed and quantified starting from the third passage helper virus stock, thus confirming the presence of SRCAs. Electronic micrographs corroborated the presence of these particles. Later, PCR analysis allowed the identification and semi-quantification the presence of the E1 region in the helper virus stocks. Conclusion: The propagation of helper virus in HEK293 cells generated SRCAs that could easily be identified by means of different cellular and molecular methodologies. E-mail: [email protected] P 188 Transcriptional effect of steroid drugs on the CMV promoter of an adenoviral vector Francisco Matinez-F 1; Alejandro Oceguera-C 2; Maria Luisa Sandoval-R 2; Hugo E Sandoval-Z 2; Hilda Villegas Castrejon 2; Catalina Machuca-R 3 1National Institute of Rehabilitation, Molecular Biotherapeutic Program, Department of Molecular and Cellular Morphology, Mexico D.F., Mexico; 2National Institute of Rehabilitation, Molecular Biotherapeutic Program, Calzada Mexico Xochimilco 289, Mexico D.F., Mexico; 3Fes-zaragoza. UNAM, Molecular Therapy, Mexico D.F., Mexico

Introduction: A sequence derived from the CMV promoter is the most common enhancer used for transcriptional control of vectors employed for gene therapy strategies. However, the interaction between pharmacological drugs and adenoviral vectors, has not been described for our adenoviral vectors potentially employed for osteosarcoma cells.

ESGCT 2008 POSTER PRESENTATIONS Objective: Evaluate the transcriptional effect of several steroid drugs in two grades of osteosarcoma cells, infected with an adenoviral vector with CMV promoter. Material and Methods: An adenoviral vector was generated with the ORF of luciferase gene under transcriptional control of the CMV promoter for in vivo grade. 5 x105 MCG1 and MCG3 cells, were grown in standard cell culture protocol. Cells were infected with 10, 25, 50 and 100 MOI’s to determine the optimal infection. Before the drug interaction, all cells were starved from serum for 24 hrs. The five steroid drugs (beta estradiol, testosterone, betamethasone, hydrocortisone, and dexamethasone), were used in serial concentrations and 2% of FBS. The protein extractions were performed at 3 and 6 hours and finally submitted for luciferase assay. All experiments were performed in triplicate. Results: In general, the luciferase activity, was decreasing gradually according to the grade of malignancy. The reference of the basal activity was obtained for myoblast cells. The average of response of the several drugs in MCG3 is the 50% to the MCG1 (aver  3000 cl/m). Estradiol and testosterone have no significant effect on the promoter. However, dexamethasone and hydrocortisone increased 30 and 45% respectively, the level expression at 3 and 6 hours. Interestingly, we found an inhibitory effect of betamethasone 35% over the basal activity (2900 LC/s). All drugs show a negative transcriptional effect in MCG3, on average a 50% minus to the control (4000 LC/s). Specifically, beta estradiol and hydrocortisone reached only 10 and 15% of the basal expression. E-mail: [email protected] P 189 Influence of lyophilisation and reconstitution of aqueous plasmid DNA solutions and long term stability Markus Blaesen ; M Schmeer ; R Baier ; M Schleef PlasmidFactory GmbH & Co. KG, Bielefeld, Germany Physical and chemical stability of plasmid DNA is a requirement for the development of DNA-based pharmaceuticals capable of being stored, shipped and applied even under critical environmental conditions. The influences of different factors on the lyophilisation process of aqueous plasmid DNA solutions were examined with two model plasmids: the 4472 bp plasmid pBfeGMCSF (PF138) and the 6233 bp reporter gene vector pCMV-luc (PF461). The filling volume of the micro tubes depends on the DNA concentration to provide a total amount of 100 g pDNA per vial: in case of 0.5 mg/mL a volume of 200 L, in case of 1.0 mg/mL a volume of 100 L and in case of 5.0 mg/mL 20 L were chosen. The plasmids were dissolved in saline, TE buffer or WFI. Changes in the topology distribution after lyophilisation and reconstitution were measured densitometrically after agarose gel electrophoresis and more accurately with the capillary gel electrophoresis technology (CGE). The latter method allows identification of the most important plasmid topologies like ccc-monomer, ccc-dimer, oc-forms and linear forms. A slight shift to the open circular form could be observed. In most cases the specification of 95% ccc forms was fulfilled after reconstitution. The examinations within the long term stability of aqueous plasmid DNA solutions went on with the above mentioned plasmids. The results were also obtained by densitometry and capillary gel electrophoresis. After nearly 2 years of storage at 20°C pBfeGMCSF–independent from the

ESGCT 2008 POSTER PRESENTATIONS DNA concentration, buffer conditions have remained stable. The distribution of the main topologic forms was unchanged. At 4°C PF138 shows a shift from the ccc-forms of the monomer and dimers to the oc-forms. The results achieved from the reporter gene vector were very similar. E-mail: [email protected]

1169 P 191 Silencing of HIV-1 with RNA interference: A multiple shRNA approach Olivier ter Brake ; Karin J. von Eije ; Westerink Jan-Tinus ; Nick C. Schopman ; Ben Berkhout Academic Medical Center, Univ. of Amsterdam, Laboratory of Experimental Virology, Meibergdreef 15 1105 AZ, Amsterdam, Netherlands

P 190 RNAi-based therapy to target TAK1 in rheumatoid arthritis Gabriel Courties 1; Virginia Seiffart 2; Virginie Escriou 3; Daniel Scherman 3; Andrea Hoffmann 2; Christian Jorgensen 4; Gerhard Gross 2; Florence Apparailly 4 1Inserm,

Inserm U844, Université Montpellier 1, Montpellier, France; 2Helmholtz-Centre for Infection Research, Signalling and Gene Regulation, Braunschweig, Germany; 3Inserm, Inserm U640, CNRS, UMR8151, ENSCP, Paris, France; 4Inserm, Inserm U844, UM1, CHU Montpellier, Montpellier, France Background: Rheumatoid arthritis (RA) is the most frequent rheumatic condition characterized by chronic inflammation leading to joint degradation. While current biotherapies have revolutionized RA treatment, they still have draw-backs supporting the need for the development of alternative strategies. Transforming growth factor-beta activated kinase 1 (TAK1), a downstream mediator of IL1 and TNF signal pathways, plays a central role in the regulation of catabolic events and inflammatory processes. We thus investigated the feasibility of targeting the TAK1 by RNA interference (RNAi) in arthritis to reduce both joint inflammation and degradation. mediated inflammatory cascades Methods: TAK1 siRNA sequences were validated in vitro on the macrophage-cell line J774.1, assessing the protein levels by western blotting and by downregulation of p38- and JNK-activation after TNF challenge. For in vivo administration, 10g of siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA, and injected intravenously in DBA/1 mice having collagen-induced arthritis (CIA). Clinical course of the disease was assessed by paw thickness over time and histological scores were obtained at euthanasia. The immunological balance was assessed using anti-type II collagen (bCII) assays, measuring the bCII-specific T cell proliferation, and quantifying cytokine levels in sera and knee-conditioned media by ELISA. Results: The TAK1 siRNA sequence reproducibly silenced at least 50% of the protein expression compared with a control siRNA, and the downstream signalling pathway. Weekly intravenous injections of anti-TAK1 siRNAlipoplexes significantly reduced incidence and severity of established CIA compared with the control siRNA lipoplex-injected group. The clinical effect was associated with a decreased secretion of IL-6, IFN- and TNF- both locally (knee joints and draining lymph nodes) and systemically (blood and spleen). Conclusion: The innovative liposome-based siRNA formulation provides an efficient tool for screening new factors involved in RA and RNAi-based gene therapy targeting TAK1 might be a promising strategy to interfere with inflammation and bone homeostasis, two important features in RA. E-mail: [email protected]

Double-stranded RNA, expressed in the cell as short hairpin RNA (shRNA), can induce gene silencing via a process known as RNAi. Previously, we have shown that stable expression of a shRNA against HIV-1 strongly inhibits HIV-1 replication in vitro. However, a single shRNA is not sufficient to maintain inhibition. One of the hallmarks of RNAi, its sequence specificity, presents a way out for the virus, as single nucleotide substitutions in the target region can abolish the inhibition. For the development of a durable gene therapy that prevents such viral escape, we combine multiple shRNA inhibitors that target highly conserved regions in the HIV-1 RNA genome. Such a strategy is analogous to HAART in which several antivirals are used in a single treatment. When we combined four shRNAs in a single lentiviral vector, viral escape was prevented, confirming the great potential of RNAi as an antiviral approach for HIV-1.1 We also tested safety and efficacy of RNAi in the human immune system (Rag-2 / c / ) mouse model with a single shRNA vector.2 We showed that an antiviral RNAi based gene therapy on blood stem cells results in a normal immune system development. Importantly, in mature human T cells derived from these mice, HIV-1 replication was inhibited in a sequence-specific manner. For the translation of a combinatorial approach into a clinical application, selection of the most effective and safe shRNAs is required. In addition, lentiviral vector design and production needs to be optimized to obtain the high titers that are required for a clinical application. Our latest results on shRNA selection and lentiviral vector optimization will be presented. (1) ter Brake O, et al. Lentiviral vector design for multiple shRNA expression and durable HIV-1 inhibition. Mol Ther 2008 Mar;16(3):557-564. (2) ter Brake O, et al. Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2 / c / ) mouse model. Gene Ther 2008 AOP Jul 31. E-mail: [email protected] P 192 Evaluation of safety and efficacy of anti-HIV-1 shRNA expression in vivo and vitro Karin J. von Eije 1; Olivier ter Brake 1; Mireille Centlivre 1; Nicolas Legrand 2; Kees Weijer 2; Bianca Blom 2; John J. Rossi 3; Ben Berkhout 1 1Academic Medical Center, University of Amsterdam, Department of Experimental Virology, Center for Infection and Immunity Amsterdam, Amsterdam, Netherlands; 2Academic Medical Center, University of Amsterdam, Department of Cell Biology & Histology, Amsterdam, Netherlands; 3Beckman Research Institute of City of Hope, Division of Molecular Biology, Duarte, United States

Double-stranded RNA expressed as short hairpin RNA (shRNA) can induce gene silencing through a process known as RNA interference (RNAi). Previously, we have shown that stable expression of anti-HIV-1 shRNAs with lentiviral vec-

1170 tors results in strong inhibition of virus replication. However, the virus evolves to become RNAi resistant. Targeting highly conserved regions restricts the viral escape possibilities and the virus mutants that are selected closely resemble natural variation in HIV-1 sequences (1). Viral escape can be prevented by the use of a combination of shRNAs (2). For the clinical application of RNAi, transduction of hematopoietic stem cells is desirable, which will generate a constant supply of HIV-resistant cells. To test the feasibility of such an approach, we used CD34 precursor cells with a lentiviral vector encoding an anti-HIV-1 shRNA targeting the Nef region. These cells were engrafted in Rag2( / )gamma(c)( / ) mice that allow development of a human immune system. Development and maturation of the human immune system was not affected in the shNef-engrafted mice (cell numbers, cell types). After establishment of the immune system in vivo, we challenged the human CD4 T cells from these mice with HIV-1 ex vivo (3). Potent sequence specific inhibition of HIV1 was scored. In addition, in HIV-1 infected cell cultures we measured preferential outgrowth of the shRNA-expressing cells over untreated cells, a promising result for our gene therapy approach. Our latest results from in vivo and in vitro experiments in which we evaluate safety and efficacy will be presented. 1 von Eije KJ, ter Brake O, Berkhout B. Human immunodeficiency virus type 1 escape is restricted when conserved genome sequences are targeted by RNA interference. J Virol 2008 Mar;82(6):2895-2903. 2 ter Brake O, ‘t Hooft K, Liu YP, Centlivre M, von Eije KJ, Berkhout B. Lentiviral vector design for multiple shRNA expression and durable HIV-1 Inhibition. Mol Ther 2008 Mar;16(3):557-564. 3 ter Brake O, Legrand N, von Eije KJ, Centlivre M, Spits H, Weijer K, et al. Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag2( / )(c)( / )) mouse model. Gene Ther 2008 Jul 31. E-mail: [email protected] P 193 siRNA Magnetofection in vitro Olga Mykhaylyk 1; Dialekti Vlaskou 1; Edelburga Hammerschmid 1; Olivier Zelphati 2; Joseph Resenecker 3; Christian Plank 1 1Klinikum

rechts der Isar TU München, Experimental Oncology, München, Germany; 2OZ Biosceinces, Parc Scientifique et Technologique de Luminy BP13, Marseille, France; 3Kinderklinik und Poliklinik, Forschungszentrum, München, Germany Although efficient non-viral delivery systems are available, optimal siRNA delivery, in vitro and particularly in vivo, remains a challenge. Since the first report on siRNA delivery by magnetofection in 2003 [1], the potential of the method to efficiently deliver siRNA to living cells cultivated in vitro has been demonstrated in several recent publications. We have developed magnetic nanomaterials that are suitable to associate with siRNA, either alone or in combination with cationic polymers or cationic lipids used as enhancers for delivery to cultured cells upon application of a magnetic gradient field. We have also developed the procedures for examining siRNA incorporation into magnetic complexes, for evaluating their magnetic responsiveness, composition of the complexes and for characterizing their association with and uptake into cells. While a high association of siRNA delivery vectors with target cells is achieved with standard, non-magnetic vectors,

ESGCT 2008 POSTER PRESENTATIONS magnetofection leads to improved uptake into cells. This may be the primary reason why magnetofection yields highly improved dose-response profiles compared with nonmagnetic vectors for siRNA delivery [2, 3] in cultured epithelial, endothelial and other tested cell types. The developed methods [4] can be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized siRNA transduction by magnetofection in any cell type. The support through the FP6-LIFESCIHEALTH Project no. 005213, German Ministry of Education and Research, Nanobiotechnology grants 13N8186 as well as by 13N8538 German Excellence Initiative via the “Nanosystems Initiative Munich (NIM)” are gratefully acknowledged. 1. Plank C et al. Expert Opin Biol Ther (2003) 3:745-758. 2. Mykhaylyk, O. et al (2007) J. Magn Magn Mater, 311, 275281. 3. Mykhaylyk O. et al. Current Opinion in Molecular Therapeutics, in press (2008). 4. Mykhaylyk O. et al. Meth Mol Biol, Protocol article in press (2008), editor Mouldy Sioud. E-mail: [email protected] P 194 Modulation of cardiac Ca2 homeostasis by adenovector-mediated and doxycyline-inducible expression of a phospholamban shRNA embedded into a microRNA environment Tobias Grossl 1; Lennart Suckau 1; Xiaomin Wang 1; Jens Kurreck 2; Wolfgang Poller 1; Roland Vetter 3; Henry Fechner 1 1Charité

- Universitätsmedizin Berlin, CBF, Dept. of Cardiology and Pneumology, Hindenburgdamm 30, Berlin, Germany; 2Universität Stuttgart, Institut für Industrielle Genetik, Stuttgart, Germany; 3Charité - Universitätsmedizin Berlin, CBF, Institute of Clinical Pharmacology and Toxicology, Garystr. 5, Berlin, Germany

Background: Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum (SR) Ca2 pump (SERCA2a) contributes to cardiac dysfunction in chronic heart failure. Recently, we have shown that vector-mediated expression of PLB-shRNAs under the control of a constitutive U6 promoter results in highly efficient and specific PLB gene silencing in primary neonatal rat cardiac myocyte cultures (CMC). This was associated with a marked increase of the SERCA2a-catalyzed SR Ca2 sequestration. Because vector-mediated shRNA expression in vivo can lead to undesirable side effects, we have developed a novel doxycyline (Dox)-regulated adenoviral vector (AdV) system allowing drug-dependent control of PLB-shRNA expression. Material and Methods: The essential components of the Tet-On gene expression system were inserted within one AdV genome. The key components of the new vector AdR4miPLBr include the second generation Dox-dependent transactivator rtTA-M2, the Dox-dependent response promoter Tight1 and a PLB-shRNA embedded into a miR-155 environment (miPLBr). Functional effects of this new vector were analyzed in CMC. Results: Application of AdR4-miPLBr (Dox) resulted in efficient silencing of PLB mRNA and protein expression in CMC. The silencing activity was either similar or even higher in comparison to a control vector expressing a conventional shPLBr under the control of a constitutive U6 promoter. However, AdR4-miPLBr-mediated PLB silencing was also observed in the absence of Dox. This was prevented by co-

ESGCT 2008 POSTER PRESENTATIONS expression of a tetracycline-controlled transcriptional silencer (tTS). PLB silencing due to treatment of CMC with AdR4-miPLBr/tTS (Dox) resulted in a marked increase of the reticular SERCA2a-catalyzed Ca2 uptake rate. This functional effect was not observed in the absence of Dox. Conclusion: Thus, Dox induced AdR4-miPLBr mediated expression of shPLBr in a miR-155 environment enables efficient PLB knock-down which is functionally linked to improved SERCA2a-catalyzed Ca2 uptake into the SR of cultured neonatal rat cardiomyocytes. E-mail: [email protected] P 195 Optimizing active RNA trans-splicing molecules for the treatment of cystic fibrosis Pamina Schlager 1; Lloyd G. Mitchell 2; Helmut Hintner 1; Johann W. Bauer 1 1SALK

and Paracelsus Medical University, Department of Dermatology, Division of Molecular Dermatology, Salzburg, Austria; 2Retrotherapy, Retrotherapy, Bethesda, United States

Cystic fibrosis is one of the most common hereditary diseases. It is caused by a mutation in the cystic fibrosis conductance regulator (CFTR) gene resulting in a malfunctioning chloride channel. Usually a full-length gene is delivered into a “diseased” cell in order to correct the impaired protein expression, harbouring the possibility of unregulated overexpression. Trans-splicing is a technology to “reprogram” the sequence of endogenous mRNAs circumventing these problems. For this purpose we have established a FACS based high-throughput screen for the CFTR gene. With this screening system we have improved repair of the CFTR gene building reprogramming molecules (RTM) for correction of exons 5 to 24, making the molecule attractive for over 85% of the CF patients. This way we have identified RTM’s with a trans-splicing efficiency of up to 68%. The RTM’s have been introduced into CFPAC-1 cells, a pancreatic duct cell line that is homozygous for the F508del mutation, to show endogenous repair. To verify the activity of our trans-splicing molecules the functionality of the protein was analysed by protein detection assays. Transfer into an adenoviral vector and evaluation in an animal model are the next planned steps. E-mail: [email protected] P 196 Highly efficient RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors Thomas Schaser 1; Lydia Duerner 1; Barbara Schnierle 2; Klaus Cichutek 1; Christian J. Buchholz 1 1Paul-Ehrlich-Institut, Division of Medical Biotechnology, Langen, Germany; 2Paul-Ehrlich-Institut, Division of Virology, Langen, Germany

Background: RNA interference (RNAi)-mediated gene suppression represents a powerful technology in functional genomics and biomedical drug research. In cancer research, an RNAi transfer system is desirable that can be efficiently applied in vivo; i.e. will distribute siRNA to all tumour cells upon systemic administration. We are developing viral vectors based on replication-competent murine leukaemia virus

1171 (MLV). Previously, we have shown that shRNA expression cassettes are functionally active when inserted into the MLV genome (1). We have now engineered second generation vectors by flanking shRNA with microRNA-adapted sequences (miR30). Methods & Results: To provide proof of principle, expression cassettes for miR30-shRNAs targeting GFP or luciferase were inserted into the genome of MLV resulting in vectors MLV-antiGFP and MLV-antiLuc. Gene suppression was monitored at different multiplicities of infection (MOI) of the fibrosarcoma cell line HT1080 stably transduced with a single copy of the enhanced green fluorescent protein (EGFP) (HT1080-GFP) or the firefly luciferase gene (HT1080Luc). At an MOI of 1, luciferase activity was reduced by more than 90% within four days but remained unaffected when control vectors were applied. In general, silencing of the luciferase gene was more rapid. Moreover, the kinetics of gene silencing, i.e. kinetic of shRNA spreading in cell culture, was MOI-dependent. However, even at an MOI of 0.001 the maximal extend of luciferase activity downregulation could be reached. To evaluate in vivo activities, HT1080-Luc cells were infected with MLV-antiLuc and mixed with uninfected HT1080-Luc at a ratio of 1:1000. The cell mixture was then transplanted subcutaneously into NOD-SCID mice. Luciferase expression was reduced to 10% (  4%) 18 days after transplantation. Conclusion: Thus, second generation MLVs form a novel RNAi transfer system allowing longterm expression and efficient delivery and distribution of shRNAs to tumour cells in vitro and in vivo combined with a strong silencing activity. 1. Sliva and Schnierle. 2006. Virology 351:218-25. E-mail: [email protected] P 197 Specific small interfering RNAs targeted to CXCR4 and CCR5 confer HIV-1 resistance Su Jin Seo ; In Young Oh ; Tae Gyun Kim ; Hyun Song ; Min Joung Choi ; Youn Ju Park Korea Food & Drug Administration, Division Gene Therapy Products, Biologics Bureau, Seoul, Republic of Korea The recently discovered phenomenon of RNA interference(RNAi) offers another novel approach, which appears to be even more potent in targeted gene silencing and therefore can be potentially harnessed for HIV gene therapy. Infection of a susceptible cell by HIV-1 required viral envelope interactions with the primary cell surface receptor, CD4, and a coreceptor, either CXCR4 or CCR5. Therefore, a potentially promising strategy is to exploit siRNAs to prevent viral entry at the cell surface by down-regulating essential cell surface HIV-1 coreceptors. In our studies, we designed specific siRNA oligonucleotide and establishment of short hairpin RNA(shRNA) expressed plasmid constructs against HIV-1 cell surface coreceptors, CXCR4 and CCR5, to inhibit viral entry. These siRNA oligonucleotides prepared using synthetic RNA duplexes consisting of two unmodified 21mer oligonucleotides annealed together to from siRNAs for CXCR4 or CCR5. These plasmid constructs were designed to high-level express shRNA under the control of U6 promoter that employs RNA polymeraseIII (PolIII) in mammalian cells. shRNAs are synthesized from either CXCR4 or CCR5 DNA template. The resulting shRNAs are believed to be processed by Dicer to generate small RNA duplexes that

1172 resemble the active siRNA duplexes. HeLa-TZM cells transfected with siRNA oligonuceotide or shRNA expressed plasmid constructs showed significant down-regulation of their respective coreceptors. Also, we establishment of optimal condition for each siRNA oligonucleotide and shRNA expressed plasmid construct transfection and confirmed effective reduction of CXCR4, CCR5 expression levels by each coreceptor specific siRNA. Real-time PCR and Western blot analysis showed the reduced levels of intracellular synthesis of CXCR4, CCR5 and FACS analysis confirmed marked down-regulation of CXCR4, CCR5 on the cell surface. Specially, effective reduced levels of CXCR4 and CCR5 expression at 5nM siRNA concentration and 48 hours after transfection. These results showed possible to introduce promising siRNA for in vivo gene therapeutic applications. E-mail: [email protected] P 198 The GTSG.org database: A monitoring system for insertion sites of gene therapy vectors used in clinical gene therapy studies Stephanie Laufs 1; Frank A. Giordano 2; Jens Uwe Appelt 3; Ingo Roeder 4; Agnes Hotz-Wagenblatt 5; Gerhard Opelz 3; W. Jens Zeller 2; Heike Allgayer 1; Stefan Fruehauf 6 1German

Cancer Research Center, Molecular Oncology of Solid Tumors, INF 280, Heidelberg, Germany; 2German Cancer Research Center, Pharmacology of Cancer Treatment, INF 280, Heidelberg, Germany; 3University of Heidelberg, Department of Transplantation Immunology, Heidelberg, Germany; 4University of Leipzig, IMISE, Leipzig, Germany; 5German Cancer Research Center, HUSAR bioinformatics group, INF 280, Heidelberg, Germany; 6Center for Tumor Diagnostics and Therapy, Paracelsus Hospital Osnabrueck, Osnabrueck, Germany Background: Hematopoietic stem cell (HSC) gene therapy has proven to be a progressive approach in the treatment of hereditary diseases such as ADA-SCID, X-SCID, or CGD. Nevertheless, leukemia development following retroviral HSC gene therapy pointed out the demand for clinical and preclinical monitoring systems for future HSC gene therapy trials. These systems must contain 1) monitoring of vector insertion site profiles, 2) tracing kinetics of individual transplanted HSCs, and 3) cross-matching of insertion and clonal data to disease progression/regression, clinical status and outcome. Methods: Our group developed the GTSG.org database, which contains more than 20,000 gene therapy vector insertion sites and allows for high-throughput insertion site analysis. In a first run we analyzed 14,500 insertion sites and developed virus specific integration profiles of different viruses used in gene therapy trials (ASLV, EIAV, FIV, FV, HIV, MLV, SIV) in different cell types (human and rhesus CD34, T cells, HeLa, PBMC, SupT3). We analyzed insertion frequency on chromosomes, in fragile sites, genes, cancer genes, transcription factor binding sites, CpG islands, and repetitive elements. All data obtained from experimental analyses was then compared to a set of 1,000,000 random integrations (“random set”) and significance was determined using the Kolmogorov-Smirnov method. Results: We discovered that genes of T cells, PBMC, and CD34 are highly attractive targets for lentiviral vectors rather than for MLV or FV vectors. MLV based vectors

ESGCT 2008 POSTER PRESENTATIONS were seen preferentially integrating close to TSS, CpG islands, and transcription factor binding sites. We also saw an up to six-fold increased frequency of HIV, MLV and SIV insertions in cancer genes. Besides the interest to further characterize gene therapy vector insertion sites at a genomic level, this is a first attempt to perform comprehensive studies which are still lacking and which help to better estimate the risk of cancer- or tumor suppressor gene insertions in gene therapy. E-mail: [email protected]

P 199 Automated analysis of integration sites obtained after transplantation of transduced hematopoietic cells in mice Martijn H. Brugman ; Stefan Bartels ; Tobias Mätzig ; Olga Kustikova ; Ute Modlich ; Christopher Baum Hannover Medical School, Experimental Hematology, Hannover, Germany Since the discovery of insertional oncogenesis in mice and later in humans treated with gene modified hematopoetic cells, gene therapy safety research has focussed on analysis of integration sites. Identification of the vector flanking genomic regions has been performed in preclinical mouse model and in human gene therapy trials, which showed the preference for expressed genes and potential or established proto-oncogenes. To streamline integration site analysis, we developed programs that perform sequence trimming, BLAST alignment, annotation and storage of the retrieved sites. These programs were designed to perform analysis on a scale which makes them suitable to work with the output generated with high-throughput sequencing methodology (400.000 integrations), a task that would not be feasible to do manually. In addition to analysis of the genes closest to the vector integrations, we also looked at genes within  150kb, since we have shown that insertional dysregulation can influence genes further than 100 kb away and can act in the presence of intervening genes. We analyzed data generated with the different retroviral, self-inactivating retroviral and lentiviral vectors developed in our laboratory. These data were obtained by conventional insertion site mapping after long-term observation of mouse experiments, using vectors carrying fluorescent markers or functional genes. Our focus was on dominant clones as suggested by the abundance of the LM-PCR product. We looked for differences in integration profile, focusing on surrounding gene function, transcription factor binding site and integration orientation. No major single parameter was identified that distinguished between the insertion profiles of SIN and LTR gamma-retroviral vectors. In contrast, lentiviral insertions showed an unexpectedly high frequency of hits in repeat regions. As a quality control, we were able to confirm the typical TSS distance difference between retroviral and lentiviral vectors. In conclusion, we have established software for automated integration site analysis and validated it using integration sites obtained from long-term transplanted mice. An important next step would be the comparative analysis of integromes of freshly transduced and long-term transplanted samples E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 200 Ex vivo cell culture and cell-intrinsic heterogeneity play an important role in the development of clonal dominance after retroviral gene transfer into hematopoietic stem cells Olga Kustikova ; Bernhard Schiedlmeier ; Martijn Brugman ; Zhixiong Li ; Axel Schambach ; Christopher Baum Hannover Medical School, Experimental Hematology, Hannover, Germany The development of clonal imbalance after transplantation of genetically modified hematopoietic cells is a cause of concern in the long-term follow-up of patients undergoing gene therapy for the treatment of severe or acquired hematopoietic disorders. We and others have previously described how insertional proto-oncogene dysregulation by transgene integration may provoke clonal restriction and leukemia, thus becoming a dose-limiting toxicity of gene therapy. When targeting populations enriched for or depleted from hematopoietic stem cells (HSC) in the C57Bl6 CD45 chimerism model, we found that intrinsic stem cell potential is a conditio sine qua non for the establishment of expanding insertional mutants. Targeting HSC-enriched cell populations (short-term repopulating HSC, LSK CD34 Flt3-, or longterm repopulating HSC, LSK CD34-Flt3-), a comparison of gamma-retroviral transduction conditions in a 5 days culture period and lentiviral transduction in a 20h protocol revealed that the latter conditions significantly improved chimerism with a greatly increased clonal diversity in the first 8 weeks of repopulation. However, after lentiviral transduction clonal dominance progressively developed over an observation time of 6 months, although there was no evidence for insertional proto-oncogene upregulation as the underlying cause even when using a lentiviral vector with a strong internal enhancer-promoter capable of insertional long-distance effects. This points to a major role of cell-intrinsic properties in the emergence of dominant, gene-marked clones. Clonal restriction developing in the long-term follow-up after transplantation of gene-modified hematopoietic stem cells thus is not necessarily a side effect of insertional mutagenesis, but may also reflect classical “gene marking” of a stem cell clone with a strong intrinsic potential for competitive dominance. E-mail: [email protected] P 201 Factors to consider in gene therapy biodistribution studies Sarah Sheridan ; Alison Armstrong BioReliance Ltd, Research and Development, Glasgow, United Kingdom Genetic vaccine technology has become an important strategy for the development of therapies for currently untreatable diseases. Biodistribution studies are essential to address regulatory concerns and provide an important part of the safety evaluation of these novel gene therapy technologies. Biodistribution studies are designed to investigate integration or expression of the vector construct in the germ line, the persistence of the vector in the target tissue and dissemination to non-target tissues. Animal inoculation and subsequent harvesting of tissues at defined time points for

1173 detection of the vector in question by Q-PCR is an accepted procedure for a biodistribution study. BioReliance is a world leader in developing the procedures for quantitative PCR assays in biosafety testing. The high throughput nature and exquisite level of sensitivity of quantitative PCR provides the “gold standard” to meet the increasingly stringent regulatory guidelines. BioReliance uses a combination of highly robust multi-well quantitative PCR technology and high-throughput nucleic acid extraction automated systems to analyse tissue samples. QPCR analysis is conducted in accordance with regulatory guidelines (FDA). We can design customised biodistribution studies and provide all aspects of the service, from study design, assay development, through to animal inoculation, testing and fully audited final report. E-mail: [email protected] P 202 Adenovirus-mediated kallistatin gene transfer ameliorates disease progression in a rat model of osteoarthritis induced by anterior cruciate ligament transection Jenglong Hsieh 1; Ai-Li Shiau 2; Chao-Liang Wu 3; PoChuan Shen 4 1Chung Hwa University of Medical Technology, Department of Nursing, Tainan, Taiwan; 2National Cheng Kung University Medical College, Department of Microbiology and Immunology, Tainan, Taiwan; 3National Cheng Kung University Medical College, Department of Biochemistry and Molecular Biology, Tainan, Taiwan; 4Tainan Hospital, Department of Orthopedic Surgery, Tainan, Taiwan

Objective: In osteoarthritis (OA), inflammation and apoptosis are two important factors contributing to disease progression. As kallistatin can suppress inflammatory responses and reduce cell apoptosis, we investigated the therapeutic effect of kallistatin gene transfer in the rat model of OA by anterior cruciate ligament-transection (ACLT). Methods: OA was induced in Wistar rats by ACLT in the knee of one hindlimb. Adenoviral vector encoding human kallistatin (AdHKBP) was injected intraarticularly into the knee joints after ACLT. The transgene expression and inflammatory responses were determined by immunoblot analysis, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. The therapeutic effect of kallistatin was examined by gross morphology. The apoptosis of chondrocytes was quantified by TUNEL assay. Furthermore, the effects of kallistatin in combination with hyaluronic acid (HA) on the medial femoral condyles and synovia were also assessed histologically. Results: The expression of human kallistatin after intraarticular injection was identified. Kallistatin gene transfer reduced the levels of interleukin-1 (IL-1) and tumor necorsis factor- (TNF-) in joints. Examination of gross morphology revealed that rats treated with AdHKBP had reduced severity of OA compared with the control rats treated with adenoviral vector encoding green fluorescent protein (AdGFP). The protective effect of kallistatin on cartilage was accompanied by a decrease in apoptotic cells. Intraarticular administration of AdHKBP, when in conjunction with HA, significantly improved histologic scores in the knee joint according to the Mankin and synovitis grading scale. Conclusions: Local administration of adenoviral vectors encoding kallistatin significantly suppressed OA progression, accompanied by reduction of inflammatory response

1174 and apoptosis. Our results suggest that kallistatin gene therapy may be a potential treatment for OA. E-mail: [email protected] P 203 Design of phosphorodiamidate morpholino oligomers (PMOs) for the induction of exon skipping of the human DMD gene Linda Popplewell ; Capucine Trollet ; George Dickson ; Ian Graham Royal Holloway - University of London, School of Biological Sciences, Egham, Surrey, United Kingdom Background: Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations of the human DMD gene. Antisense oligonucleotides (AOs) have previously been used to skip additional exons which border the deletions, such that the reading frame is restored and internally-truncated, but functional, dystrophin expressed. AO-induced exon skipping is a future hope as a therapy for DMD. The first step to a clinical trial is the choice of the optimal AO target site for skipping of those DMD exons most commonly deleted in DMD. Methods: Overlapping PMOs were designed to exons 44, 45, 46, 51, and 53 of the human DMD gene using the following information: putative SR protein binding domains as predicted by ESEfinder, Rescue ESE and PESX analyses of exon sequence; predicted open secondary structures of the RNA as determined by the m-fold program; sequences accessible to binding as determined by hybridization analyses; previously published work. The ability of each PMO to produce exon skipping was tested in vitro in normal human skeletal muscle cells. Harvested RNA was subjected to nested RT-PCR, and skipping efficiencies determined by quantification of the PCR products by densitometry using GeneTools software. Results: Retrospective analysis of design parameters used and PMO variables revealed that active PMOs were longer (p  0.017), bound to their targets more strongly (p  0.001), had their target sites closer to the acceptor splice site (p  0.004), overlapped with predicted areas of open conformation, as defined by hybridization analysis (p  0.003), or by RNA secondary structure prediction (p  0.025), and could interfere with the binding of certain SR proteins (p  0.026). Linear discriminant analysis was performed on all possible combinations of PMO parameters and design tools that showed significance and revealed that length, SF2/ASF (BRCA1) overlap and hybridization peak overlap were the strongest predictors of PMO bioactivity. Conclusion: No design tool is strong enough in isolation, and there is no substitute for empirical analysis. However this study has highlighted the potential of using a combination of significant PMO parameters/design tools as a powerful aid in the design of bioactive AOs. E-mail: [email protected] P 204 Mdx diaphragm muscle as a target of dystrophin gene therapy Tomohiro Suga 1; Masatoshi Ishizaki 1; En Kimura 1; Katsuhisa Uchino 1; Tatsuya Koide 1; Yuji Uchida 2; Yasushi Maeda 1; Sheng Li 3; Jeffrey Chamberlain 3; Makoto Uchino 1

ESGCT 2008 POSTER PRESENTATIONS 1Kumamoto University, Neurology, Kumamoto, Japan; 2Sojo University, Pharmacology, Kumamoto, Japan; 3University of Washington, Neurology, Seattle, United States

Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder, and there is currently no effective treatment. DMD causes respiratory or cardiac failure and results in death at about 20 years of age. Especially respiratory insufficiency is a major problem in the management of DMD patients. In mdx mouse, a DMD model, the pathological feature of diaphragm is severely affected, which is similar to the skeletal muscle of human DMD . Although, the muscle strength assay of the dystrophic diaphragm has been used to estimate mdxmouse respiratory impairment, systemic functional assessments compared with histopathological analysis have not been demonstrated. Here we report a sensitive procedure using whole-body plethysmography to monitor respiratory parameters detected during early insufficiency in the mdx mouse. In result, the dystrophic changes in the diaphragm lead to respiratory dysfunctions. Moreover, we propose gene therapy approaches targeting diaphragm using the viral vectors. These total assessment system, including whole-body plethysmography, may be useful to evaluate the therapeutic approaches for the neuromuscular disease models. E-mail: [email protected] P 205 Co-administration of AAV8 myostatin propeptide and codon optimised microdystrophin in MDX model of duchenne’s muscular dystrophy Keith Foster 1; Ian Graham 1; Helen Foster 1; Capucine Trollet 1; Paul Yaworsky 2; Frank Walsh 2; Paul Sharp 3; Dominic Wells 3; George Dickson 1 1Royal

Holloway, University of London, School of Biological Sciences, Egham, United Kingdom; 2Wyetn Research, Clinical Discovery, Collegeville, United States; 3Imperial College London, Cellular and Molecular Neuroscience, London, United Kingdom Duchenne’s muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1/3500 male births. DMD is caused by the lack of dystrophin in the skeletal muscle. Lack of dystrophin within the muscle leads to muscle fibres which are highly prone to exercise induced injury, which consequently results in progressive rounds of degeneration and regeneration of the muscle, leading to respiratory or cardiac failure in the third decade of life. Use of myostatin propetide has been shown to improve pathology in mdx mice. Here we evaluate whether a gene therapeutic approach of myostatin inhibition leads to improvement in the pathophysiology of the dystrophic phenotype in the mdx female mouse. AAV8 myostatin propeptide was administered by tail vein injection (4  10E11 vg in 100l) and AAV8 microdystrophin was administered by intramuscular injection (5x10E10 vg in 25l). There is no significant difference in the whole body mass between control and treated groups, but codon optimised microdystrophin normalised individual TA muscle mass to C57Bl10 controls and were significantly smaller than myostatin propeptide treated mdx and mdx controls. Microdystrophin treatment, irrespective of myostatin propeptide administration, resulted in a greater specific force compared to mdx, although still significantly lower than C57Bl10 controls. Interestingly, irrespective of myostatin propeptide adminis-

ESGCT 2008 POSTER PRESENTATIONS tration, resistance to eccentric contractions was normalised to C57Bl10 controls following microdystrophin administration, however myostatin propeptide treatment alone was significantly reduced compared to mdx controls. E-mail: [email protected] P 206 Comparison of AAV8 and AAV9 microdystrophin as therapeutic vectors for the treatment of Duchenne’s muscular dystrophy Helen Foster ; Keith Foster ; Takis Athanasopoulos ; Capucine Trollet ; George Dickson Royal Holloway, University of London, School of Biological Sciences, Egham, United Kingdom Duchenne’s muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1/3500 male births. DMD is caused by a lack of dystrophin protein in skeletal muscle. Lack of dystrophin compromises the integrity of the muscle cell membrane and results in progressive rounds of degeneration and regeneration of the muscle leading to the replacement of muscle fibres with non contractile fibrotic tissue and fatty infiltrates. Gene therapy strategies for the delivery of dystrophin to skeletal muscle have been hampered by a number of factors. AAV mediated strategies for gene transfer of dystrophin to muscle have been limited by the small cloning capacity of AAV vectors. As a result smaller versions of the gene or microdystrophins have been designed based on mutations observed in the milder allelic disorder Becker Muscular Dystrophy (BMD). Very efficient gene transfer of dystrophin is crucial if restoration of muscle function is to be achieved. Recently this has been achieved by systemic administration of AAV based vectors, but this has been at prohibitively high doses. We have previously demonstrated that codon optimisation of microdystrophin results in a significant improvement in the expression level of microdystrophin following intramuscular plasmid electrotransfer and systemic administration of a relatively low dose (3x1011vg) of AAV8 codon optimised microdystrophin. The aim of the current study was to further investigate the potential of AAV vectors to deliver microdystrophin efficiently to the skeletal muscle following a low systemic dose. Data will be presented on the comparison between AAV8 and 9 serotypes to deliver codon optimised microdystrophin. Additional data will be presented on the potential re-administration of AAV9 microdystrophin to further allow lower individual doses of AAV microdystrophin to be delivered to dystrophic muscle. E-mail: [email protected] P 207 Optimising efficacy of morpholino antisense oligonucleotide induced exon-skipping in vitro in the mdx mouse Caroline McCulley ; Dominic Wells Imperial College London, Neuroscience and Mental Health, London, United Kingdom Background: Duchenne muscular dystrophy (DMD) is a lethal muscle degenerative disorder resulting from a loss of dystrophin expression. As in-frame mutations in the dystrophin gene are associated with the milder Becker form of muscular dystrophy, antisense therapy has been used to at-

1175 tempt to correct the reading frame in human and mouse muscle, both in vitro and in vivo. Antisense oligonucleotides (AON) can modify splicing of the primary transcript by targeting the exon splicing enhancers or the 3/5 splice sites. Given the large number of mutations spread across this huge gene, many different AONs will be required to treat patients. Potential efficacy of candidate AONs is most easily tested with in vitro systems. However, some antisense molecules such as the phosphorodiamidate morpholino oligomers (PMOs) are uncharged and as such are not easily administered to cells in culture except as complexes involving a charged oligonucleotide leash and a lipid transfection reagent. However, naked PMOs are quite efficient in vivo. Methods: In order to develop a testing method that uses the naked PMO we used a morpholino AON (MD23D(7-18) PMO) that targets the 5 donor splice site of intron 23 in the mdx mouse model of DMD. Using primary mdx myoblasts, a comparison was made between administration of PMO alone, lipofection of leashed PMO, and nucleofection of PMO. Results: Our findings indicate that the use of nucleofection to administer MD23D(7-18) PMO results in the most effective exon skipping in primary mdx myoblasts. Conclusion: We therefore propose that nucleofection is the method of choice for screening the action of PMOs in vitro. E-mail: [email protected] P 208 A novel muscle stem cell line for assessing non-viral gene therapy Sofia Muses 1; Jenny Morgan 2; Dominic Wells

3

1Imperial

College London, Cellular and Molecular Neuroscience, London, United Kingdom; 2Institute of Child Health, UCL, Dubowitz Neuromuscular Unit, London, United Kingdom; 3Imperial College London, Cellular and Molecular Neuroscience, London, United Kingdom Background: This study characterised a new immortalised satellite cell derived cell-line, H2K 2B4. The cell-line will be used to investigate non viral gene therapy, using Class II DNA transposons. Methods: The H2K 2B4 clone was generated from a single satellite cell; it is a conditional immortal cell-line which proliferates and differentiates under selective conditions. H2K 2B4 cells differentiation ability was evaluated in vitro and in vivo, along with their ability to be transfected. Results: A four-day in vitro differentiation assay showed the expected expression pattern of the myogenic markers: Desmin, Pax 7, MyoD, Myogenin and Fast/Slow Myosin. On transplantation into irradiated mdx nu /nu mouse muscle, the H2K 2B4 cells were able to stably generate muscle as well as contribute to the satellite cell population. No tumours were detected. H2K 2B4 cells can be transfected by Nucleofection or transduced by retrovirus with both methods achieving high transfection efficiencies. However, when using Lipofectamine 2000 reagent the transfection efficiency is limited. We hypothesised that the peri-cellular matrix surrounding the cells may hinder transfection efficiency. To investigate this, H2K 2B4 cells were first treated with bovine hyaluronidase (an enzyme which digests the hyaluronan in the peri-cellular matrix), and then transfected using Lipofectamine 2000 reagent. Higher transfection efficiencies were observed in cells treated with bovine hyaluronidase. This approach also enhances transfection efficiency in C2C12 cells. Conclusion: Overall the H2K 2B4 cell line is a good representation of a muscle stem cell model in vitro and in vivo.

1176

P 209 The combined application of deflazacort and cyclosporin A enhances utrophin expression in skeletal muscle cells of DMD patients in cell culture Stefanie Grunwald 1; Arpad von Moers 2; Ina Koch 3; Astrid Speer 1 1TFH-Berlin,

Molecular and Cell Biology, Berlin, Germany; clinical centre Spandau, Paediatric clinic, Berlin, Germany; 3TFH-Berlin, Bioinformatics, Berlin, Germany 2Westend

Background: Duchenne muscular dystrophy (DMD) is one of the most frequently inherited neuromuscular diseases in children. It is caused by the absence of dystrophin, which influences several down-stream signal transduction pathways. Recently we summerised them in a complex bioinformatic network (Grunwald et al. 2008). Theoretical knock-out studies in the network highlighted calcineurin/NFATc signalling as the most relevant part. The phosphatase calcineurin activates the nuclear factor of activated T-cells (NFATc) leading to transcription of certain target genes, e.g. utrophin A (UTRNA), myogenic factor 5 (MYF5), and cyclin dependent kinase p21. UTRNA is homologous to dystrophin. MYF5 and p21 regulate differentiation and proliferation. Methods: Here we present the influence of deflazacort and cyclosporin A (CsA) used in DMD therapy on mRNA and protein expression of the NFATc target genes and skeletal muscle cells (SkMCs) proliferation. Results: Deflazacort and CsA are proposed to act antagonisticly on calcineurin and thereby NFATc. The separate use of deflazacort and CsA led to an increased proliferation of SkMCs without affecting the expression of MYF5, and p21 on mRNA level. The combined application showed normal proliferation. UTRNA mRNA and protein expression was not changed by CsA. But enhanced values were observed when using deflazacort. This effect was increased by concurrent application of both. On mRNA level an increase of three- to sevenfold was observed and of about twofold on protein level. The effect on UTRNA expression seems to be restricted to SkMCs of DMD patients. Conclusion: Our data suggest that deflazacort and CsA separately used, can positively influence SkMCs proliferation of normal persons as well DMD patients in cell culture. However, the combination of both abolishes this effect but increases the UTRNA expression on mRNA and protein level for SkMCs of DMD patients. Both outcomes support the positive impact on DMD patients as observed in clinical applications. E-mail: [email protected]

Background: Dystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophies. While progress have been made on vector development, allowing sustained expression of therapeutic genes, it is still difficult to assay the restoration of the muscle function from small biopsies. Methods: Here we used atomic force microscopy (AFM) to probe specifically the dystrophin-mediated physical connection of the sarcolemma with the cytoskeleton of live myofibers, using small tissue explants. Results: The lack of dystrophin was shown to affect the elasticity of the sarcolemma of individual fibers within muscle tissues from young mice. This provided a very sensitive and quantitative assay of the properties of normal and dystrophic myofibers, before symptoms have developed. The rescue of dystrophin expression by AAV vector-mediated exon skipping, or the ectopic expression of utrophin by in vivo electroporation, normalized the elasticity properties of dystrophic muscles as probed by AFM. The restoration of normal muscle elasticity values was commensurate to the functional recovery of whole muscle strength. AFM thus allowed an estimation of the percentile of functionally rescued myofibers and of the extent of the corrective benefits of these gene transfer approaches. Conclusions: Atomic force microscopy provides a sensitive quantification of the physical and functional recovery of myofibers within small tissue explants, and it may complement current measurements of whole muscle strength that lack single fiber resolution. AFM may therefore provide a method of choice to assay the therapeutic success of gene or cell-based approaches of muscular dystrophies. Because this method probes individual cells within a tissue context, it may also be of wider use to assess therapies relying on the expression of other cellular or extracellular structural proteins.

W IT HD RA W N

E-mail: [email protected]

P 210

AB ST RA CT

H2K 2B4 cells can be easily transfected making them a suitable model for non viral gene therapy. We are currently using a Class II DNA transposon, Sleeping Beauty, to investigate stable integration of transgenes into the genome of H2K 2B4 cells.

ESGCT 2008 POSTER PRESENTATIONS

E-mail: [email protected]

P 211 Importance of checking exon skipping events prior to clinical trials using systemically delivered antisense morpholino in Duchenne muscular dystrophy patients Kimura Shigemi 1; Shiho Nakano 1; Kowshi Yoshioka 1; Isao Fujii 2 1Kumamoto University Graduate School, Department of Child Development, Kumamoto, Japan; 2Sojo University, Faculty of Pharmaceutical Sciences, Kumamoto, Japan

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities of the dystrophin gene. DMD, which manifests into a severe muscle weakness phenotype, results from an out-of-frame deletion(s) in the dystrophin gene. In contrast, BMD, which results from an in-frame deletion(s) in the dystrophin gene, causes a milder muscle weakness. Antisense-mediated exon skipping, which changes out-of-frame deletions to in-frame ones, is a very promising therapeutic approach for DMD. The target exon is 51, the exon that when excluded, is predictive of allowing the restoration of the dystrophin open reading frame (ORF) in 17% of patients with DMD caused

ESGCT 2008 POSTER PRESENTATIONS by a deletion (deletions of exons 45-50, 47-50, 48-50, 49-50, 50, 52, and 52-63). These approaches have been shown to be successful in mice and dog animal models of DMD. Prior to the initiation of clinical treatment, it is very important to confirm the correct exon skipping events. In this study, we developed a system that can easily identify patients who are eligible for the therapy. Fibroblasts from DMD patients with a 48-50 exon deletion of the dystrophin gene were isolated, induced to differentiate to the myogenic lineage by AdMyoD (adenoviral vectors encoding MyoD regulated by CAG Promoter) and transfected with antisense morpholino B30, which is located at the splicing enhancer and/or I25 which is located at exon donor. The skipping event of exon 51 was correctly detected using B30 but not while using I25 (the band size was longer than the correct size). Sequence analysis showed that the fragment included only part of exon 51. Interestingly, while using both B30 and I25, we detected the correct band only. We conclude that it is paramount that the exon-skipping event is correctly performed prior to initiating clincial treatment. This detection should utilize two or more antisense mechanisms to ensure stable exon-skipping. E-mail: [email protected]

1177 crosis, which was at later times followed by extensive muscle regeneration. Conclusions: This study demonstrates that intramuscular mIL-12 gene electrotransfer resulted in efficient transfection of muscle tissue and systemic distribution of mIL-12. The treatment had a pronounced antitumor effect in solid subcutaneous tumors as well as on lung metastases of murine sarcomas. E-mail: [email protected] P 213 Lentiviral-based gene therapy for oculopharyngeal muscular dystrophy Olivia Bales 1; Rafael Yanez 1; Gillian Talbot 2; Capucine Trollet 3; George Dickson 4; Silvere van der Maarel 5; Michael Antoniou 1 1King’s

College London, Medical and Molecular Genetics, London, United Kingdom; 2King’s College London, Medical and Molecular Genetics, London, United Kingdom; 3Royal Holloway, School of Biological Sciences, London, United Kingdom; 4Royal Holloway, School of Biological Sciences, London, United Kingdom; 5Leiden University Medical Center, Department of Human Genetics, Leiden, Netherlands

P 212 Intramuscular interleukin-12 gene electrotransfer results in antitumor and antimetastatic effect in two murine sarcoma tumor models Tevz Gregor 1; Coer Andrej 2; Kranjc Simona 1; Mesojednik Suzana 1; Kamensek Urska 1; Cemazar Maja 1; Krzan Mojca 2; Sersa Gregor 1 1Institute

of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia; 2University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia

Background: Interleukin-12 (IL-12) is a cytokine that stimulates immune system and also has direct antitumor effects. The aim of our study was to evaluate the antitumor efficacy of intramuscular mIL-12 gene electrotransfer in induced lung metastases and subcutaneous tumors of two murine sarcomas, SA-1 and LPB. Methods: Lung metastases and subcutaneous tumors were induced by intravenous or subcutaneous injection of tumor cells, respectively. Electrotransfer of mIL-12 was performed by intramuscular injection of plasmid DNA followed by local application of electric pulses. Mice bearing subcutaneous tumors were treated by mIL-12 gene electrotransfer 3-times every second day. Treatment effectiveness was evaluated by tumor growth delay assay. Mice with induced lung metastases were treated 24 h before (prophylactic treatment) or at three different time points after the injection of cancer cells. 8 or 14 days post-treatment, mice were sacrificed and the number of metastases determined. In addition, total mIL12 and IFN- in serum were determined by ELISA. Histological changes of treated muscle tissue were also examined. Results: The growth of subcutaneous tumors treated by mIL-12 gene electrotransfer was delayed for 90 days in SA1 and 41 days in LPB tumors and 28 % of SA-1 and 13% of LPB tumor complete responses were observed. The number of lung metastases was significantly reduced after electrotransfer of mIL-12; for 90 % in SA-1 and 92 % in LPB tumors. The serum mIL-12 and IFN- levels increased up to 200-fold compared to base-line levels after four administrations of mIL-12 gene electrotransfer. The transfected muscle tissue was infiltrated by immune cells and muscle fiber ne-

Oculopharyngeal muscular dystrophy (OPMD) is a lateonset autosomal dominant disorder with symptoms including eyelid drooping and swallowing difficulties. OPMD is caused by small expansions of 2-7 residues of a polyalanine tract at the N-terminus of poly(A) binding protein nuclear 1 (PABPN1) that leads to protein aggregation and pathologically-linked nuclear inclusion body formation. We are exploring gene therapy strategies for OPMD using muscle-specific expression from lentiviral vectors. An intracellular antibody (“intrabody”) has been identified that prevents formation of PABPN1 nuclear aggregates, and we have used lentiviral vectors to express this under the control of the muscle-specific human desmin (DES) promoter/enhancer in mouse myoblasts, including a primary skeletal muscle cell model of OPMD. We were unable to achieve good expression of the intrabody in this system, primarily due to the poor transduction efficiency of mouse myoblasts with lentiviral vectors. We have now generated a human cell model of OPMD, using lentiviral vectors to express wild-type and mutant PABPN1 under the DES promoter/enhancer in the immortalised human myogenic cell line Hu5/E18. E18 cells can be very efficiently transduced by lentivirus, so this should prove to be a better model system for testing the expression levels of our lentiviral vectors, and determining the effect on aggregation of PABPN1. We will use this system to test the intrabody virus, alongside another lentivirus expressing shRNA against PABPN1. We also plan to test these therapeutic vectors in OPMD myoblasts isolated from patients. E-mail: [email protected] P 214 Lentiviral vectors transduce human skeletal myoblasts with a far greater efficiency than murine myoblasts Olivia Bales 1; Pascal Leclere 2; Marie Lecomte 2; George Dickson 3; Michael Antoniou 2 1King’s College London, Medical and Molecular Genetics, London, United Kingdom; 2King’s College London, Medical and Molecular Genetics, London, United Kingdom; 3Royal

1178 Holloway, School of Biological Sciences, London, United Kingdom Lentiviral vectors (LVs) have great potential for gene therapy of muscle diseases such as Duchenne Muscular Dystrophy (DMD), as they can integrate stably into dividing and non-dividing cells, and have a relatively large transgene capacity. LVs offer the potential for long-term stable expression of therapeutic genes in myogenic cells and differentiated muscle fibres. So far, most LVs designed for muscle gene therapy have been tested in murine myoblast culture or whole animal models. We show here that the transduction efficiency of LVs into human myoblast/myofibre cultures is far greater compared to murine equivalent cells, a finding with important implications for the field of muscle gene therapy with this vector system. Transduction efficiency of the murine myoblast line C2C12 and ImmortoMouse-derived immortalised primary murine myoblasts was compared to primary human myoblasts and the immortalised human myogenic cell line Hu5/E18. We used LVs carrying three different promoterGFP expression cassettes; the ubiquitous promoters SFFV (spleen focus forming virus) and A2UCOE (ubiquitous chromatin opening element from the human HNRPA2B1CBX3 locus) and the muscle-specific promoter/enhancer region from human desmin gene (DES). The different cell types were transduced at the same MOI for each virus. FACS analysis in each case showed that the percentage of GFP-positive cells was significantly higher in human cells compared to mouse, with an average increase of 40-fold. Planned future experiments include demonstrating this difference in transduction efficiency in a therapeutic context. We have constructed an LV carrying a micro-dystrophin gene under DES promoter/enhancer control and will use this to transduce immortalised human DMD myoblasts and mdx mouse myoblasts to compare the extent to which dystrophin expression can be rescued in each cell type. E-mail: [email protected] P 215 Evaluation of tumorigenic effect of bcl-2 overexpressed in transplanted mioblast Alejandro Oceguera-C 1; Hugo E Sandoval-Z 1; Maria Luisa Sandoval-R 1; Ernedira G Estrada-V 2; Catalina Machuca-R 3; Alejandro Zentella-D 4; Hilda Villegas-C 1; Francisco Martinez-F 1 1National

Institute of Rehabilitation, Molecular Biotherapeutic Program, Mexico, DF, Mexico; 2National Institute of Rehabilitation, Department of Pathology, Mexico, DF, Mexico; 3FES-Zaragoza. UNAM, Molecular Therapy, Mexico, DF, Mexico; 4IIB-UNAM/INCMNSZ, Biochemestry, Mexico, DF, Mexico Introduction: The Bcl-2 overexpression has been studied for cytoprotection in several kind of cells and tissues. Previously we found, than the myoblasts transformed with a retroviral vector to overexpressing bcl-2 protein in model of muscular reparation, results in cytoarchitecture preservation and an enhancement of myotubes diferentiation. However, the retroviral transformation, provides too a potential tumorigenic factor. Objective: To evaluate the tumorigenic effect of the repared muscle with transformed myoblast cells with a retroviral vector to overexpressing bcl-2 in a in vivo model. Materials and Methods The following groups (n  4) of

ESGCT 2008 POSTER PRESENTATIONS nude mice were transplanted with 2  106 cells. Group A with normal cells (control); Group B, myoblasts trasformed with a retrovirus vector based on MLV system with CMVGFP cassette (GFP group); and Group C, myoblasts transformed with retrovirus containing Bcl-2 (under Pol I promoter). All animals were maintained in the animal care facilities, under controlled environment and normal diet. After 4 months, animals were sacrificed and studied for molecular and histological analysis. Results: Animals of the control group show macroscopical rates of fibrosis and atrophy. One animal shows a tumour around the mouth. The GFP group shows an increased tumor growth in the site of transplant, and one tumour on the shoulder. The Bcl-2 group shows similar size with the contralateral leg. The molecular analysis of RT-PCR shows a minor expression of bcl-2 levels related to the GFP expression. The histological analysis was similar to histological patterns obtained during the day 30. The data shows that the effect of bcl-2 could improve the control of diferentiation and possibly its implication on the growth process of myoblasts. Aditionally, this conserved effect could be a consequence of the different promoter, suggesting that the low levels of bcl-2 in muscle improves its biological effect and a tumor phenotype negative. E-mail: [email protected] P 216 Cardiac mesoangioblasts are committed, self-renewable progenitors, associated with small vessels of both adult normal and cardiomyopathic mouse ventricle Stefania Crippa 1; Marco Cassano 1; Carlos Claver Clavel 1; Claudia Altomare 2; Daniela Galli 3; Laura Perani 4; Graziella Messina 4; Antonio Zaza 5; Giulio Cossu 4; Maurilio Sampaolesi 1 1University

Hospital Gasthuisberg, KULeuven, Interdipartimental Stem Cell Institute, Leuven, Belgium; 2Università degli studi di Milano Bicocca, Dipartimento Biotecnologie e Bioscienze Milano, Milano, Belgium; 3University of Pavia, Human Anatomy, Pavia, Italy; 4DIBIT, HSR, Stem Cell Research Institute, Milan, Italy; 5Università degli studi di Milano Bicocca, Dipartimento Biotecnologie e Bioscienze Milano, Milan, Italy Background: On the basis of distinct cell surface markers, different types of cardiac stem-like cells have been isolated that are able to restore cardiac function after ischemic injury with variable efficacy. None of these cells shows spontaneous cardiac differentiation. Our recent work on mesoangioblasts isolated from post-natal skeletal muscle indicated that these cells, possibly because of a local commitment, exhibit efficient differentiation into skeletal muscle. On this basis, we isolated mesoangioblast-like cells from different regions of the adult heart of both wild type and cardiomyopathic mice. Methods: Hearts were collected from four weeks-old beta sarcoglycan KO (bSG KO) and wild-type (WT) mice and dissected into small fragments. Cardiac mesoangioblasts (cMabs) appeared like small round and poorly adhering cells after 7-10 days, they could cloned by limiting dilution. The selected clones were analyzed in terms of surface antigens and tissue specific markers expression. We studied their ability to differentiate in all mesodermal cell lines with particular interest in their myogenic and cardiomyogenic potential, In vivo cMabs were transplanted both in the tibialis anterior muscles and in the infarcted ventricles.

ESGCT 2008 POSTER PRESENTATIONS Results: WT cMabs express many cardiac transcription factors and spontaneously differentiate into beating cardiomyocytes that assemble mature sarcomeres and express cardiac ion channels. When injected into the ventricle after coronary artery ligation, cardiac mesoangioblasts efficiently generate new myocardium in the border of the necrotic zone. bSG KO cMabs although express the majority of cardiac transcription factors they also express MyoD and Myf5, and differentiate spontaneously into myotubes. If injected in tibialis anterior muscles of both cardiotoxin-treated and dystrophic muscle dystrophic cMabs differentiate in regenerating muscle fibers. Conclusion: In our work we highlighted a possible role of beta sarcoglycan protein in signal transduction pathways for cell differentiation. The cardiac tissue degeneration in bSG KO mice could be explained, at least in part, for the lack of cardiac differentiation of local committed progenitor cells. E-mail: [email protected] P 217 Cardiac dystrophic mesoangioblasts: Molecular characterization of in vitro and in vivo differentiation Mattia Quattrocelli 1; Stefania Crippa 1; Marco Cassano 1; Matilde Bongio 2; Flavio Ronzoni 2; Ilaria Perini 1; Maurilio Sampaolesi 1 1K.U.Leuven,

Translational Cardiomyology, Stem Cell Institute, Leuven, Belgium; 2University of Pavia, DiMeS, Human Anatomy, Pavia, Italy In muscular dystrophies a cardiac involvement represents a complication leading the patients to a more dramatic worsening of health condition. Limb-girdle muscular dystrophy type 2E (LGMD 2E) is caused by mutations in the sarcoglycan gene. The expression of the sarcoglycan complex is necessary for the stabilization of dystrophin at the sarcolemma level: if this link is interrupted, skeletal and cardiac myofibres degenerate. Recently, we showed that a class of vessel associated stem cells, named mesoangioblasts, isolated from muscle biopsies of mice [1], dogs [2] and humans [3] can regenerate skeletal fibres in dystrophic subjects. Moreover, wild-type cardiac mesoangioblasts can efficiently differentiate in vitro an in vivo into cardiomyocytes [4]. We isolated and characterized cardiac mesoangioblasts (cdMABs) from heart biopsies of a mouse model of LGMD 2E. In particular, we evaluated the expression of surface markers and cardiac transcription factors and we tested the ability to migrate into heart and filter organs, after intravenous delivery into cardiomyopathic mice. Despite their ability to home and differentiate into cardiomyocytes, cdMABs fail to regenerate necrotic areas of the myocardium. By real time expression array, their transmigration and transdifferentiation ability seems affected by signal-transduction pathways, such as Notch and Wnt signalling, that drive cell fate. Exploring the balancing patterns of these molecular signals will probably shed new light on myogenic program and dystrophy therapy. [1] Sampaolesi M et al, Cell therapy of alpha sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts, Science 2003 Jul 25; 301(5632): 487-92 [2] Sampaolesi M, Blot S et al, Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs, Nature 2006 Nov 30; 444(7119): 574-9 [3] Dellavalle A, Sampaolesi M et al, Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells, Nat Cell Biol 2007 Mar; 9(3): 255-267

1179 [4] Galvez BG, Sampaolesi M et al, Cardiac mesoangioblasts are committed, self-renewable progenitors, associated with small vessels of juvenile mouse ventricle, Cell Death Differ 2008 May 23 E-mail: [email protected]

P 218 Constructing a biological pacemaker from cardiac progenitor stem cells Gerard Boink 1; Joost Sluijter 2; Arie Verkerk 1; Diane Bakker 1; Shirley van Amersfoorth 1; Teun de Boer 3; Marcel van der Heyden 3; Toon van Veen 3; Marie-José Goumans 2; Jurgen Seppen 4; Jacques de Bakker 1; Hanno Tan 1 1Academic Medical Center, Experimental Cardiology, Heart Failure Research Center, Amsterdam, Netherlands; 2University Medical Center Utrecht, Laboratory of Experimental Cardiology, Division of Heart & Lungs, Utrecht, Netherlands; 3University Medical Center Utrecht, Department of Medical Physiology, Division of Heart & Lungs, Utrecht, Netherlands; 4Academic Medical Center, AMC Liver Center, Amsterdam, Netherlands

Background: Reaching a physiologic beating rate remains a major challenge in current biopacemaker development. A promising approach to increase beating rate is the use of stem cells that are both differentiated into pacemaker-like cells and additionally modified with a pacemaker gene. We have previously demonstrated that human cardiac progenitor cells (CPCs) can be efficiently differentiated into spontaneously beating aggregates. Here we studied the effects of lentiviral overexpression of the pacemaker gene HCN4 in undifferentiated CPCs co-cultured with neonatal rat cardiac myocytes (NRCMs). Methods: Human CPCs were isolated from fetal hearts obtained after elected abortion. CPCs were isolated from the cardiac cell suspension using magnetic beads coated with a Sca-1 antibody. CPCs were subsequently cultured and transduced in non-differentiating conditions. The effect of lentiviral HCN4 overexpression was studied in single undifferentiated CPCs using the perforated patch-clamp technique. Spontaneous beating was measured in cultures of HCN4 or GFP overexpressing undifferentiated CPCs co-cultured with NRCMs. Results: FACS analysis demonstrated that 98.8% of the CPCs could be transduced at a MOI of 50. While pacemaker current (If) was virtually absent in non-transduced CPCs (n  4), lentiviral HCN4 overexpression resulted in a large hyperpolarization-activated, time-dependent inward current ( 21 pA/pF at 140 mV, n  8) with properties typical of If . Half maximal activation voltage and slope factor of steady-state activation were 89.6  2.8 and 8.2  1.2 mV (n  8), respectively. In hybrid cultures, spontaneous beating was increased from 32  6 (n  4; GFP-CPC-NRCMs) to 85  14 beats per minute (n  4; HCN4-GFP-CPC-NRCMs; P  0.005). Conclusion: Human CPCs are efficiently transduced by lentiviral vectors. HCN4 overexpressing CPCs increased spontaneous beating of hybrid cultures, which also demonstrated electrical coupling between the two cell populations. These findings may aid the development of autologous regenerative therapies including the biological pacemaker. E-mail: [email protected]

1180

ESGCT 2008 POSTER PRESENTATIONS

P 219 Constitutive expression of HIF-1 and HIF-2 in bone marrow stromal cells differentially promote their proangiogenic properties Jeremy Ben-Shoshan 1; Shulamit Schwartz 2; Galia Luboshits 1; Sofia Maysel-Auslender 1; Aya Barzelay 1; Sylvie Polak-Charcon 3; Iris Barshack 3; Eldad Tzahor 4; Adiel Barak 5; Hani Levkovitch-Verbin 6; Gad Keren 1; Jacob George 1 1Sourasky Tel-Aviv Medical Center, Cardiology, 6 Weizmann St, Tel-Aviv, Israel; 2Assaf Harofeh Medical Center, Ophthalmology, Zrifin, Israel; 3Sheba Medical Center, Pathology, Tel-Hashomer, Israel; 4Weizmann Institute, Biological Regulation, Rehovot, Israel; 5Sourasky Tel-Aviv Medical Center, Ophtalmology, Tel-Aviv, Israel; 6Sheba Medical Center, Ophtalmology, Tel-Hashomer, Israel

Background: Bone marrow stromal cells (BMSCs) contain endothelial progenitors capable to participate in postnatal angiogenesis. However, the limited effectiveness of these cells as a therapeutic tool so far, has prompted the need to ameliorate their angiogenic properties. We explored the capacity of hypoxia inducible factors (HIFs), the main regulators of the cellular response to hypoxia, to improve BMSCs angiogenic potential. Methods: BMSCs grown in endothelial-inducing conditions were retroviraly transduced to express stable forms of HIF-1 and HIF-2. Cells were then characterized by their ability to bind ac-LDL and Ulex europaeus-Lectin and to express CD45, CD11b, CD29, CD90, Flk-1, CD31 and Tie-2. BMSCs were functionally assessed for adhesion to fibronectin and mature endothelium as well as chemotaxis towards SDF1 and VEGF and tube formation on Matrigel. Paracrine evaluation was based on expression of angiogenic factors and protein arrays of mature endothelial cells (ECs) stimulated by medium collected from BMSCs. Eventually; we performed in vivo BMSCs systemic delivery using a corneal micropocket neovascularization model. Results: HIF-1 and, to a greater extent, HIF-2 promoted the differentiation of BMSCs toward endothelial lineage, as evident by markers expression and improved adhesive properties. Whereas chemotaxis towards SDF-1 was higher in both HIF1/2-BMSCs, enhanced migration towards VEGF was found only in HIF-2-BMSCs, consequent by increased expression of Flk-1 receptor. HIF- stabilization was also associated with upregulation of angiogenic factors and improved tubulogenesis. Mature ECs stimulated by HIF--BMSCs exhibited further angiogenic activation and improved ICMA-1/VCAM-1-mediated adhesion, compared to controls. Eventually, delivery of HIF-BMSCs, prominently HIF-2, significantly improved cells homing and overall neovascularization. Conclusion: Our results support the use of HIF- genes, particularly HIF-2, to induce a broad pro-angiogenic program in BMSCs. We also point at the importance of the paracrine effects induced by BMSCs, which must be considered during the implementation of future cell-based angiogenic therapy. E-mail: [email protected] P 220 Specific and long-term genetic engineering of adult stem cells in the rodent brain by peptide-tagged Ad vectors Anke Schmidt 1; Anja Stoll 1; Jens Pahnke 2; Steve Hildebrandt 3; Andreas Wree 4; Brigitte M. Pützer

1

1University of Rostock, Department of Vectorology & Exp. Gene Therapy, Rostock, Germany; 2University of Rostock, Department of Neurology, Rostock, Germany; 3University of Rostock, Institute of Anatomy, Department of Vectorology & Exp. Gene Therapy, Rostock, Germany; 4University of Rostock, Institute of Anatomy, Rostock, Germany

Background: The intrinsic differentiation and migratory properties of stem cells in the adult brain make them an intriguing source for restorative cell replacement strategies and as delivery vehicles for therapeutic genes to diseased brain regions. Neural stem cell (NSC) development and migration is controlled by the microenvironment in neurogenic regions, suggesting that direct in situ manipulation of endogenous stem cells may be beneficial over current ex vivo-transplantation strategies. A key requirement herefore is the availability of a safe and specific gene delivery system. Methods: We recently reported selective transduction of hippocampal SC in the dentate gyrus of pNestin-GFP transgenic mice by direct injection of fluorescent-protein encoding adenoviral (Ad) vectors engineered to bind SC-specific peptides. Our data clearly referred to a stem cell heterogeneity between the hippocampus and other SC homing areas, implicating that selective vectors are needed for individual brain regions. Results: In the present study, we identified peptide ligands that specifically bind to SC isolated from the subventricular zone (SVZ) in vitro. These peptides also efficiently mediate specific binding and vector internalization in vivo after injection into the adult mouse brain. SVZ-SC specific infection was demonstrated by co-labeling of infected GFPexpressing cells with SC and proliferation markers. Moreover, we show that viruses allow to follow differentiation of infected cells, respectively their daughter cells, over a several months time period. Notably, peptide-tagged Ad vectors are brain region but not species-specific and can also be used to target human NPC. Conclusion: This approach provides the first tool for specific manipulation of endogenous adult SC and direct utilization of their potential to treat neurodegenerative and neoplastic CNS disorders originated from different brain regions. This work was supported by grants from BMBF, DFG, and the FORUN program of Rostock University Medical Faculty. E-mail: [email protected] P 221 Direct antigen presentation by leukemic dendritic cells promotes expansion of allogeneic leukemia-reactive T lymphocytes Monica Casucci 1; Serena Kimi Perna 2; Attilio Bondanza 2; Zulma Magnani 1; Massimo Bernardi 3; Alessandro Crotta 3; Alessandro Cignetti 4; Cristina Tresoldi 5; Katharina Fleischhauer 6; Federico Caligaris-Cappio 7; Fabio Ciceri 3; Claudio Bordignon 2; Chiara Bonini 2 1San

Raffaele Scientific Institute, Cancer Immunotherapy and Gene Therapy Program, Milan, Italy; 2San Raffaele Scientific Institute, Cancer Immunotherapy and Gene Therapy Program, Hematology and Bone Marrow Transplantation Unit, Milan, Italy; 3San Raffaele Scientific Institute, Hematology and Bone Marrow Transplantation Unit, Milan, Italy; 4Institute for Cancer Research and Treatment, Laboratories of Tumor Immunology, Candiolo, Turin, Italy; 5San Raffaele Scientific Institute, Department of Immunohematology-Blood Transfusion, Milan, Italy; 6San Raffaele Scientific Institute, Department of

ESGCT 2008 POSTER PRESENTATIONS Immunohematology-Blood Transfusion, San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 7San Raffaele Scientific Institute, Department of Oncology, Milan, Italy Donor lymphocyte infusion (DLI) after allo-HSCT allows curing chronic myeloid leukemia but is less effective against acute myeloid leukemia (AML). This may be due to multiple factors, among which a qualitative and quantitative defect in the antigen presenting cell (APC) compartment. Myeloid blasts are poor APC, but can be forced to differentiate into dendritic cells (DC), offering a model in which the most potent APC and the tumor cell coincides. Such leukemic dendritic cells (LDC) have the potential to boost the graft-versus-leukemia effect of DLI. Unfortunately, traditional cytokine-based protocols for LDC differentiation were successful only in a minority of primary AML (30%). Recently, a strategy based on calcium ionophore (CI) A23187IL-4 has been reported to overcome cytokine resistance of most primary AML. The aim of this study was to assess the efficacy of the CI-based protocol in differentiating primary and secondary AML, evaluating the beneficial immunostimulatory effect of LDC differentiation by a comparison between APC activity of LDC and original blasts. In a cohort of 14 AML patients, we observed that a short (48 hrs) exposure to A23187IL4 induced LDC differentiation of a large proportion (12/14) of primary and secondary AML blasts. Compared to original blasts, LDC significantly upregulate molecules of the immunological synapse (ie: CD86, CD80, HLA-DR, CD54 and CD58), maintaining the expression of surface disease markers (ie: CD34 and CD117) and the leukemic antigen WT1. This phenotypic profile correlated with a high T cells stimulatory capacity, similar to that of healthy mature DC. Most importantly, LDC were superior to the original blasts in promoting the expansion of leukemia-reactive T lymphocytes with a central memory phenotype from HLA-identical and haploidentical donors. Strikingly, we observed a clear correlation between T-cell stimulation capacity of LDC and the level and proportion of LDC differentiation and maturation. Interestingly, this protocol directly induced a mature CCR7 CD83 phenotype in 44% of LDC, with the highest frequency observed among secondary AML. The efficacy of CIbased differentiation strategy provides the rational for novel adoptive immunotherapeutic approaches to treat high risk AML. E-mail: [email protected] P 222 Critical role of iNOS in MSC-mediated immune suppression in experimental arthritis Carine Bouffi ; Claire Bony ; Gabriel Courties ; Christian Jorgensen ; Daniele Noel Inserm, U844, Montpellier, France Multipotent mesenchymal stromal cells (MSC) are adult stem cells characterized by their differentiation potential into multiple lineages and their immunosuppressive properties. This last property is used here to evaluate the efficacy of primary MSC to inhibit inflammation-associated symptoms in the murine experimental model of collagen-induced arthritis (CIA). The role of iNOS and IL-6 in the suppressive activity of MSC is investigated. Methods. MSC were isolated from DBA1 and wild type (wt), inducible nitric oxid synthase (iNOS) / or IL-6 /

C57Bl6 mice. Cells were immunophenotyped and function-

1181 ally tested. In the tumour model, MSC were subcutaneously co-injected with the B16 melanoma cells (105 of each cell type) in BALB/c mice. In CIA, 106 MSC were intravenously injected at various times after collagen II immunization of DBA1 mice. Arthritis was evaluated by the measure of paw swelling and immunological parameters. Results. All primary MSC populations were characterized by their common phenotype (CD45 , CD11b , CD44, CD73, Sca1) and differentiation potential towards chondrocytes, adipocytes, osteoblasts. However, compared to wt MSC, iNOS / or IL-6 / exhibited a highly reduced immunosuppressive effect. Using the B16 model, tumours develop in 80% of mice when wt MSCs were coinjected compared to 20 or 40% respectively, with iNOS / or IL-6 /

MSC. In the CIA model, when injected on day 18 and 24, syngeneic and allogeneic MSC were able to significantly decrease the incidence and clinical signs of arthritis. The immunological parameters confirmed a decreased inflammatory response with MSC. When iNOS / MSC were injected, the mean paw swelling did not significantly differ from that measured with wt MSC but the clinical score was dramatically and significantly increased. No significant differences were observed between IL-6 / or wt MSC. Conclusion. This study shows the efficacy of systemic injection of syngeneic or allogeneic MSC in the treatment of experimental arthritis in a restricted window of application. The role of iNOS in suppressing the inflammatory response in CIA is demonstrated whereas involvement of IL-6 is still under debate. E-mail: [email protected] P 223 Differentiation of human embryonic stem cells into alveolar epithelial type II pneumocytes Paola Spitalieri 1; Maria Favia 2; Maria Chiara Quitadamo 1; Giancarlo Cortese 3; Arianna Malgieri 1; Andrea Luchetti 1; Gennaro Citro 3; Alfonso Baldi 4; Giovanna Lazzari 5; Lorenzo Guerra 2; Valeria Casavola 2; Giuseppe Novelli 1; Federica Sangiuolo 1 1Tor

Vergata University, Department of Biopathology, Genetics Unit, Rome, Italy; 2University of Bari, Department of General and Environmental Physiology, Bari, Italy; 3Regina Elena Institute, SSD-SAFU, Rome, Italy; 4II University of Naples, Department of Biochemistry, section of Pathology, Naples, Italy; 5ISILS, Laboratory of Tecnologies of Reproduction, Cremona, Italy Background: The pluripotency of human embryonic stem cells (hES) offers a new opportunity in tissue engineering and cell therapy. The purpose of this study is to generate in vitro airway epithelial cells derived from human embryonic stem cells (HUES-3) and to inject them in vivo into bleomycin-damaged mouse to evaluate their engraftment capacity. Methods: HUES-3 (46, XY cell line) were treated with SAGM medium for 3-5-15 days. Molecular and immunohistochemical analyses were performed for evaluating the expression of surfactant protein A, B, C and aquaporin 5, specific markers of type II pneumocytes. Nude mouse was both irradiated and intratracheally injected with bleomycin for 15 days. About 500,000 SAGM-committed cells were inoculated after 3-6-10 days of treatment. Results: Real Time RT-PCR was performed on SAGMtreated and untreated HUES-3 cells after 15 days of differentiation protocol. A transcript increase of 10 and 4 fold was

1182 evidenced for two different isoforms of SP-C transcript, SPC 117 and SP-C 134. Immunohistochemical analyses confirmed SP-C expression and aquaporin-5 and CFTR localization in treated cells. Moreover HUES-3, grown on permeable supports and treated with SAGM, showed a transepithelial resistance 150/cm2. Histochemical data confirmed the damage in bleomycin-mouse lung specifically in type II pneumocytes by ematossilin-eosin and Van Gieson trichrome analyses. Results reveal a typically fibrosis area of inflammation, epithelial hyperplasia and interstitial edema. In vivo experiments focused to evaluate the engraftment capacity of SAGM-committed cells are in progress. Conclusion: Our results demonstrated the possibility to use hES for reparing damaged lung by cell therapy approach. Further examination could be important to support these data suggesting that this approach could be applied for the treatment of airway diseases, as Cystic Fibrosis (CF, OMIM: # 602421), using CF-mutated cells as target for a combined gene and cell therapy approach. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS ulation of bangslab-labelled NSC/NPC(LUC) was implanted (5  105 cells by stereotactic injection) in the CNS of immune-competent ROSA26L-S-L-Luc mice. Cell implant localisation and survival was monitored during 4 weeks by in vivo BLI and MRI, which demonstrated intrinsic survival of implanted NSC/NPC(LUC). In addition, our BLI and MRI observations were validated post-mortem by histological characterisation of the NSC/NPC(LUC) grafts. Conclusions: We here demonstrate that in vivo localization and survival of grafted NSC/NPC in the CNS of mice can be monitored by combined BLI and MRI. E-mail: [email protected] P 225 Stem cell-niche and lysosomal cathepsins Sabata Martino 1; Roberto Tiribuzi 2; Francesco D’Angelo 2; Anna C Berardi 3; Maurilio Sampaolesi 4; Angela Gritti 5; Gabriella Cusella de Angelis 4; Aldo Orlacchio 1 1University

P 224 In vivo bioluminescence and magnetic resonance imaging of neural stem/progenitor cells following implantation in the central nervous system of immunecompetent mice Kristien Reekmans 1; Nathalie de Vocht 2; Bart Tambuyzer 2; Irene Bergwerf 2; Jasmijn Daans 2; Shyama Chatterjee 2; Philippe Jorens2; Dirk Ysebaert 2; Eric Van Marck 2; Annemie Van Der Linden 3; Zwi Berneman 2; Peter Ponsaerts 2 1UA,

Medicine, Universiteitsplein 1 T1.17, Wilrijk, Belgium; Medicine, Wilrijk, Belgium; 3UA, Biomedical Science, Wilrijk, Belgium 2UA,

Aims of the Study: (I) In vitro characterisation of adherently growing primary cultures of neural stem/progenitor cells (NSC/NPC) by flow cytometry and immunocytochemistry. (II) In vivo characterisation of neural stem/progenitor cell grafts in the central nervous system (CNS) of mice by non-invasive bioluminescence imaging (BLI) and magnetic resonance imaging (MRI), and by post-mortem histological analysis. Results: * PART I: First, adherently growing NSC/NPC cultures were initiated from embryonic brains (E14) of ROSA26-L-S-L-Luciferase transgenic mice. Established cultures, which self-renew for up to 30-50 passages, were characterized by flow cytometry and found to express A2B5 and NCAM, without detectable expression of mesenchymal (Sca1, CD106), haematopoietic (c-kit, CD45) and endothelial (CD31) markers. In addition, immunocytochemical stainings demonstrated expression of the intracellular markers GFAP, BLBP and SOX2. Next, ROSA26-L-S-L-Luc mouse-derived NSC/NPC cultures were lipofected with mRNA encoding Cre-recombinase in order activate expression of the Luciferase reporter gene (70.000 fotons/s/100.000cells). Moreover, the ability of the established NSC/NPC(LUC) line to differentiate in vitro into GFAP astrocytes and Tuj1 neurons, as demonstrated by immunohistochemical stainings, further indicated the neural stem/progenitor cell identity of our cultured NSC/NPC(LUC). * PART II: Second, cultured NSC/NPC(LUC) were labelled with 0.96m fluorescent (Dragon Green) magnetite-containing micron-sized particles (BangsLab particles) and efficiency of uptake was determined by flow cytometric analysis ( 90%). Next, this pop-

of Perugia, Medicina Sperimentale e Scienze Biochimiche, Perugia, Italy; 2University of Perugia, Medicina Sperimentale e Scienze Biochimiche, Sez. di Biochimica e Biologia Molecolare, Perugia, Italy; 3Ospedale Pediatrico Bambino Gesó, IRCCS, Roma, Italy; 4Università degli Studi di Pavia, Centro Interdipartimentale di Ingegneria Tissutal, Pavia, Italy; 5TIGET, HSR, Milan, Italy Background: A stem cell niche is a specialized microenvironment where resident cells orchestrate self-renewal and differentiation decisions through molecular signalling that may or may not depend on the type of adult tissue in which they are localized. Within a niche, stem cells are physically anchored through adhesion molecules that apparently mediate cross-talk to the niche itself as well as the external milieu. Lysosomal cathepsins play crucial roles in diverse biological processes, either immuno-related as within the antigenpresentation pathway, or cancer-related as during angiogenesis, cell proliferation, apoptosis, and metastatic progression. Known for their proteolytic activity toward certain adhesion molecules such as beta-integrin and E-cadherin, these enzymes appear to be functionally linked to the biology of the stem cell niche. To elucidate their role, we investigated the expression of lysosomal Cathepsins D, S, B and L in stem cells derived from different embryonic origins: i) endodermal (hematopoietic stem cells, HSCs), ii) mesodermal (mesoangioblasts,MSCs), and iii) ectodermal (neural stem cells,NSCs). Methods: Stem cells were isolated as previous described (1-3). Cathepsins were analysed by RT Real Time PCR and Western Blotting using anti-Cathepsin-D, -S, B and L as primary antibodies. Results: Our results show that Cathepsins S, B and L are expressed as precursor forms in stem cells regardless their embryonic origin, whereas Cathepsin D, an aspartic protease, generally appeared as a mature enzyme protein. However, there was a significant difference in Cathepsin D expression in HSCs isolated from bone marrow, a hematopoietic niche, versus circulating HSCs derived from either peripheral or cord blood. In the latter, only the enzyme’s precursor form was detected. Conclusion: Together, these observations point to Cathepsin D as a dynamic player in stem cell biology and suggest that cathepsins might function as proteolytic reservoirs susceptible to specific signalling for activation from the surrounding milieu.

ESGCT 2008 POSTER PRESENTATIONS References 1-Tiribuzi R, et al. Min. Biotecnologica 2008;20: 59-67 2-Gavarez BG, et al. J Cell Biol. 2006;174:231-43 3-Gritti A, et al. J Neurosci. 2002;22:437-45 Acknowledgements: FIRB RBIP06FH7J_002 E-mail: [email protected] P 226 Extensive characterisation of hepatic progenitors derived form human embryonic stem cells Thomas Touboul 1; Sebastien Corbineau 1; Amélie Martinez 1; Helene Strick-Marchand 2; Ludovic Vallier 3; Anne Weber 1 U804, Kremlin-Bicêtre, France; 2Institut Pasteur, Unité des Cytokines et Développement Lymphoïde, Paris, France; 3CIMR, Surgery, Cambridge, United Kingdom

1Inserm,

Background: The pluripotent status of human embryonic stem cells (hESCs) confers upon them unique value for regenerative medicine, as they are capable of generating a large variety of cell types. However, generating fully functional cells from hESCs and achieving this goal using clinicallycompatible conditions remain major challenges. Modelling the early steps of embryonic development in vitro may provide the best approach for generating differentiated cells with native properties. We have developed a chemically defined culture conditions for achieving specification of hESCs into definitive endoderm and to differentiate them further into immature hepatic progenitor cells (hepatoblasts). Methods: The cells (H9, Wicell) are grown three days in presence of Activin, BMP4 and FGF2 to generate the definitive endoderm population and then hepatogenic factors are added sequentially to the medium to commit these cells towards a hepatic fate. Cell differentiation was analysed by RT-PCR, immunocytochemistry, Western Blots and FACS for cell surface and intracellular hepatic markers. HESCs-derived hepatoblasts were transplanted into the liver of NOD/SCID uPA/rag  / to assess their functionality in vivo. Results: HESCs derived endodermal cells expressed definitive endoderm markers Sox17, Goosecoid, Mixl1 and do not express the visceral endoderm marker Sox7. Early hepatoblasts coexpress AFP and CK19, HNF4, APOAII, the epithelial cell surface markers EpCam, E-cadherine. These cells express also more mature hepatocyte functions including albumin, tyrosine aminotransferase, tryptophane oxygenase, alpha-1-antitrypsin, the asialoglycoprotein receptor. Transplanted mice have been killed at 2 and 4 weeks after transplantation and their livers are presently being analysed for human hepatic functions. We have also transduced the hESCs with a lentivirus carrying the GFP under the control of APOAII promoter and purified the transduced hepatoblasts. The cells kept their pluripotency properties. Conclusion: We have established a chemically defined protocol to produce hepatic progenitors. We are now assessing the in vivo functionality of the hepatoblast population and the purified cell line. E-mail: [email protected] P 227 Ex vivo expanded autologous natural killer cells as a novel therapeutic approach in multiple myeloma Evren Alici ; Tolga Sutlu ; Bo Björkstrand ; Mari Gilljam ; Birgitta Stellan ; Hareth Nahi ; Hernan Concha Quezada ; Gösta Gahrton ; Hans-Gustaf Ljunggren ; M. Sirac Dilber

1183 Karolinska Institutet, Department of Medicine, Stockholm, Sweden Multiple myeloma (MM) is an incurable plasma cell malignancy accounting for about 2% of all cancer deaths and nearly 20% of deaths caused by hematological malignancies. Combinations of stem cell transplantation, targeted pharmacotherapy and immunotherapy constitute the most promising therapeutic options currently available. Natural killer (NK) cells are among the candidates for new means of immunotherapy; however, their potential clinical use in MM has not been extensively studied. In this study, we explored the possibility of expanding NK cells from the peripheral blood of seven newly diagnosed, untreated MM patients, using good manufacturing practice (GMP)-compliant components. By the end of the culture period, the total cell population had expanded on average 511-fold and, of these, NK cells had expanded on average 1625-fold resulting in a 65% domination of the culture. When the expanded NK cells were phenotypically compared to day 0 cells, increased expression of the activating receptors 2B4, CD8, CD16, CD27, CD226, NKG2C, NKG2D, NKp30, NKp44, NKp46 and the inhibitory receptors KIR2DL3 and LIR-1 were observed. Challenging the expanded cells ex vivo with tumor targets revealed that expanded cells provided a markedly elevated cytotoxic activity against autologous MM cells whereas neither day 0 nor day 5 cells showed more than baseline levels of cytotoxicity. Notably, no significant cytotoxicity against normal cells was observed. Dissection of the cytotoxic response by degranulation assays has revealed that NK cells were the main cytotoxic population during challenge with autologous MM cells. Based on these findings, we are now planning to initiate a phase I/II clinical trial to investigate the use of expanded NK cells as a support to autologous stem cell transplantation for the treatment of relapse. Alongside research to unravel the mechanisms responsible for the phenomena reported here is worthy of further exploration in order to verify their clinical potential. E-mail: [email protected] P 228 Neonatal hematopoietic stem cell transplantation following low-dose busulphan conditioning reverses osteopetrosis and preserves vision in oc/oc mice Carmen Flores 1; Maria Askmyr 1; Johan Holmberg 2; Mats Ehinger 3; Tord Hjalt 2; Johan Richter 4 1University of Lund, Dept of Molecular Medicine and Gene therapy, Lund, Sweden; 2University of Lund, Dept of Cell and Developmental Biology, Lund, Sweden; 3University of Lund, Dept of Pathology, Lund, Sweden; 4University of Lund, Dept of Molecular Medicine and Gene therapy, BMC A12, Lund, Sweden

Background: Infantile malignant osteopetrosis is a fatal disease caused by lack of functional osteoclasts ultimately resulting in bone marrow (BM) failure. The only curative treatment is BM transplantation but HLA-identical donors are not always available. In the majority of patients, TCIRG1, encoding a subunit of a proton pump essential for bone resorption, is mutated. The increase in bone mass results in secondary complications e.g. optic nerve compression which progress to blindness. Oc/oc mice have a deletion in tcirg1 and die around 3-4 weeks. We have previously shown that they can be rescued by neonatal ip transplantation of normal BM or genetically modified oc/oc fetal liver cells following irradiation conditioning. As irradiation of neonatal

1184 mice results in retinal degeneration we have not been able to assess the vision of oc/oc mice. Methods and Results: Here we changed the transplantation procedure towards a clinically more relevant setting using busulphan (7.5 or 15 mg/kg sc on day 18) to condition pregnant dams followed by i.v transplantation of cells (1x106 normal lineage depleted BM cells) 1 day after birth. Conditioning with Busulphan at 15 mg/kg resulted in high level engraftment in control mice but was toxic to oc/oc mice. However, when the busulphan dose was reduced to 7.5 mg/kg, 6 out of 7 oc/oc mice survived past expected lifespan with engraftment around 10% and correction of the skeletal phenotype. Busulphan did not have adverse effects on the retina and 8 weeks after transplantation both control and oc/oc mice retained their vision as assessed by a visual tracking drum test. Histopathology showed an intact multilayered retina, in contrast to the damaged retina of irradiated neonatal mice. Conclusion: Pre-conditioning with low-dose busulphan before BM transplantation leads to prolonged survival of oc/oc mice, reverses osteopetrosis and preserves vision at least up to 9 months after transplantation. E-mail: [email protected] P 229 Resident cardiac stem cells in the left atrial appendage— an untapped source Avishag Korkus ; Chaim Lotan ; Ronen Beeri Hadassah Hebrew University Medical Center, Cardiovascular Research Center, Heart Institute, Jerusalem, Israel Background: In the past few years compelling evidence has accumulated suggesting that the heart may retain some regenerative potential, in reaction to pathologic stresses. Cardiac stem cells (CSC) were demonstrated to concentrate in specialized niches, rather than be uniformly distributed throughout the heart. Inferring from other organs, niches of stem cells are likely to be found mainly in crypt-like area. Thus, we hypothesized that the left atrial appendage (LAA) is a very likely place to find stem cells. Methods: LAA tissue from mouse and rat was cut into 1to 2 mm2 pieces, washed and digested three times with trypsin and collagenase IV. The remaining tissue fragments were cultured as explants in complete explant medium (CEM). After three weeks, a layer of fibroblast like cells outgrew from the adherent explants. Undifferentiated cells that grew as self-adherent clusters-cardiospheres over this layer were characterized by specific stains for stem cell (c-kit) and cardiac progenitor cell (GATA4) markers. To assess clonality, single cells were seeded on matrigel and expanded in DMEM - Ham’s F12(1:1) medium containing growth factors. Results: The round undifferentiated cells grew in large numbers around the explant. These cells stained positive for c-kit and GATA4. Clones of up to 15 cells grew 4 days after separate seeding of a single cell. In one case, spontaneous differentiation occurred with spontaneous contraction. Conclusion: We have isolated, in substantial numbers, multiplying and clonogenic cells from LAA of mice and rats. These cells exhibit a cardiac stem cell phenotype. This preliminary study suggests that the LAA may be a source of cardiac stem cells. These may be used for potential cardiac replacement therapies, and to better understand the mechanisms driving cardiomyocytes to reenter the cell cycle. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 230 Hsv-tk suicide gene modified mesenchymal stem cells mediated tumour therapy Miroslava Matuskova 1; Andrea Pastorakova 1; Kristina Hlubinova 1; Lubica Hunakova 2; Veronika Altanerova 3; Cestmir Altaner 3; Lucia Kucerova 3 1Cancer

Research Institute, Laboratory of Molecular Oncology, Bratislava, Slovakia; 2Cancer Research Institute SAS, Laboratory of Immunology, Vlarska 7, Bratislava, Slovakia; 3Cancer Research Institute SAS, Laboratory of Molecular Oncology, Vlarska 7, Bratislava, Slovakia Background: Utility of mesenchymal stem cells in targeting the sites of tumour formation has been shown in many models recently. We have tested whether Herpes Simplexthymidine kinase (HSV-tk) expressing adipose tissue-derived mesenchymal stem cells (MSC) could be used in cancer gene therapy as suicide gene delivery vehicle. Methods: MSC, thymidine kinase expressing adipose tissue-derived MSC (TK-MSC), human glioma cell lines U-118 MG, 8-MG-BA, 42-MG-BA and ganciclovir (GCV). Direct co cultures were evaluated by viability, fluorimetric assay and flow cytometry. Results: We have shown HSV-tk expression in AT-MSC upon retrovirus transduction. We studied the effect of TKMSC mediated ganciclovir conversion on glioma cells in direct co cultures in vitro. Fluorescently labelled, suicide gene transduced TK-MSC directly co cultured with glioma cells exhibited cytotoxic effect in the presence of prodrug. 8-MGBA cells appear to be most sensitive to TK-MSC mediated therapy. GCV concentration as low as 25 g/ml, being nontoxic for MSC and/or glioma cells, exhibited significant cytotoxicity of more than 50% in the presence of TK transgene on glioma cells 8-MG-BA. Cancer cells underwent apoptosis within a short period of time (48 hrs) as demonstrated by AnnexinV staining. Our data indicate gap junction formation capability between TK-MSC and glioma cells thus resulting in strong bystander effect. Conclusion: These data show that TK-MSC are able to mediate cytotoxic effect towards glioma cells and can serve as suitable vehicles for delivery prodrug converting gene to tumour mass. Based on these results we intend to start evaluation of therapeutic efficiency in vivo. E-mail: [email protected] P 231 Development of a safe and effective stem cell transplantation strategy by the use of antibody-based minimum intensity preconditioning and delayed infusion of suicide-gene transduced donor T lymphocytes in the mouse model of X-linked chronic granulomatous disease (X-CGD) Yasuo Takeuchi 1; Makoto Otsu 1; Emiko Takeuchi 2; Sachie Suzuki 1; Shin Kaneko 1; Motihito Okabe 1; Akihiro Kume 3; Masafumi Onodera 4; Hiromitsu Nakauchi 1 1Institute of Medical Science, University of Tokyo, Center for Stem Cell and Regenerative Medicine, 4-6-1 Shirokanedai, General Research Building 2F, Minato-ku, Tokyo, Japan; 2Kitasato University School of Medicine, Clinical Investigation, Sagamihara, Japan; 3Jichi Medical University, Genetic Therapeutics, Shimotsuke, Japan; 4National Res Inst for Child Health and Develop, Genetics, Tokyo, Japan

Allogeneic hematopoietic stem cell transplantation (Allo HSCT) is useful for treatment of life-threatening, non-ma-

ESGCT 2008 POSTER PRESENTATIONS

1185

lignant diseases. The procedures, however, inevitably involve substantial risks including 1) conditioning toxicity, 2) graft rejection and 3) graft versus host disease (GVHD). To develop a safe and effective transplantation strategy for clinical application, we have designed a combinatorial method consisting of anti-CD40L monoclonal antibody (aCD40L mAb)-based minimum conditioning and delayed infusion of suicide-gene transduced donor T lymphocytes in murine HSCT models. We used C57BL/6  C3H F1 mice (BCF1) as donors and C57BL/6  DBA2 F1 mice (BDF1) as recipients to mimic haplo-identical allo-HSCT model. Low dose irradiation (3 Gy) on day -1 followed by aCD40L mAb injection (2 mg) and donor bone marrow cell infusion (2  107) on day 0, reproducibly led to induction of stable mixed chimerism (45.2  12.5 % B cell donor chimerism at 12 wks post-BMT) in treated mice (12 of 12). Next, we injected donor T cells (1  107) into mixed chimeric mice. They showed subsequent enhancement of donor cell chimerism, also having exhibited some symptoms of GVHD. We are now in the process of testing introduction of the herpes simplex virus-thymidine kinase (HSV-TK)/GCV system into donor T lymphocytes as a fail-safe measure. Although still preliminary, these results likely indicate the possible development of a safe and effective HSCT strategy that utilizes; 1) harmless aCD40L mAbbased chimerism establishment, and 2) secure induction of full-donor chimerism with the use of donor T cells that are equipped with the fail-safe suicide system. Finally, we are now attempting this procedure for the X-CGD mouse model, which is characterized by functional defects in phagocytes. We now propose this transplantation strategy as a promising treatment option applicable for many intractable disorders including X-CGD.

(carotid, fascia, gonad, liver and skin). Based on SAGE results of BM-MSC and UCV-MSC, we chose genes hyper or hypoexpressed related to homing (SDF1, CXCR4, VCAM1 and ITGA4), osteogenesis (RUNX2 and BMP2) and immunomodulation (TGF1). Results: We observed that BM-MSC, UCV-MSC and MSC from diverse fetal tissues (FT-MSC) share similar immunophenotypic profile with differentiation capacity for osteocytes and adipocytes. In culture conditions, FT-MSC were smaller, quickly expandable and senesced later than BMMSC and UCV-MSC. Gene expression analysis by qPCR showed that SDF-1 (75,3 fold than in UCV-MSC and 28 fold than in FT-MSC) and VCAM-1 (2,4 fold than in UCVMSC and 16,8 fold than in FT-MSC) were higher in BMMSC, focusing on this important role of the bone marrow, hematopoiesis support. CXCR4 was detected in FT-MSC and UCV-MSC but, not in BM-MSC. RUNX2, known as an important initiator of osteogenesis, was higher in BM-MSC (3,5 fold than in FT-MSC), emphasizing a tissue-related function of these cells for osteogenic differentiation. At last, TGF1, a potent immunosupressor, was 2,5 fold higher in BM-MSC than in FT-MSC. The other genes selected did not show significant gene expression differences among all sources. Conclusion:Taken together, these results indicate a widely distribution of MSC, a great proliferative potential of FTMSC and suggests that these cells may have an improved migratory ability. On the other hand, these data suggest that BM-MSC may be more efficient in osteogenesis and as an immunosupressive source for cell therapy. Lastly, these data show that each source has its particular subset of genes differentially expressed.

E-mail: [email protected]

E-mail: [email protected]

P 232

P 233

Comparison between multipotent mesenchymal stromal cells (MSC) obtained from adult bone marrow, umbilical cord vein and diverse fetal tissues

Influence of genetic modification of mesenchymal stem cells on their differentiation capacity and immunogenicity

Ana Valeria Gouveia Andrade 1; Maristela Delgado Orellana 2; Simone Kashima 3; Patricia V. Bonini Palma 4; Karina Rosa Solano 2; Samia Rigotto Caruso 2; Rochele Azevedo 3; Israel Tojal 5; Aparecida Maria Fontes 6; Luiz Cesar Peres 7; Luciano Neder 7; Dimas Tadeu Covas 1

Emrah Aydogan ; Marese Cregg ; Caroline Ryan ; Mary Murphy ; Timothy O’Brien ; Frank Barry ; Thomas Ritter

1Faculty

of Medicine of Ribeirao Preto, Clinical Medicine Department, Ribeirao Preto, Brazil; 2Bloodcenter of Ribeirao Preto, Cell Therapy Laboratory, Ribeirao Preto, Brazil; 3Bloodcenter of Ribeirao Preto, Molecular Biology Laboratory, Ribeirao Preto, Brazil; 4Bloodcenter of Ribeirao Preto, Flow Cytometry Laboratory, Ribeirao Preto, Brazil; 5Bloodcenter of Ribeirao Preto, Bioinformatics Laboratory, Ribeirao Preto, Brazil; 6Bloodcenter of Ribeirao Preto, Gene Transfer Laboratory, Ribeirao Preto, Brazil; 7Faculty of Medicine of Ribeirao Preto, Pathology Department at Clinical Hospital, Ribeirao Preto, Brazil Background: Multipotent mesenchymal stromal cells (MSC) can be isolated from various adult and fetal tissues. MSC are of great interest for their potential to repair bone and cartilage, and also their immunosuppressive properties. We hypothesized that different sources of MSC have distinct proliferative potential and specific tissue-related gene expression. Methods: To explore these differences, we compared adult bone marrow MSC (BM-MSC), umbilical cord vein MSC (UCV-MSC) and MSC derived from 5 different fetal tissues

National University of Ireland Galway, Medicine, The Regenerative Medicine Institute, University Road, Orbsen Building, Galway, Ireland Background: Mesenchymal stem cells (MSCs) are multipotent stem cells with the capacity to differentiate into different lineages. Moreover they exhibit immunomodulatory properties as they can suppress the proliferation of allogenic T cells. The objective of this study was to examine the influence of retroviral and adenoviral gene transfer in rat MSCs on their differentiation capacity and immunogenicity in vitro. Methods: Bone-marrow derived rMSCs were isolated and transduced with adenoviral (Ad) and retroviral (RV, MoMuLV-based) vectors expressing the reporter gene Enhanced Green Fluorescent Protein (EGFP). Transduction efficiency and persistence of transgene expression was measured using flow cytometry. To verify the maintenance of multi-differentiation capacity of MSCs after gene transfer, transduced MSCs were induced to differentiate into adipogenic and chondrogenic lineages. Furthermore, the effect of both transduced and non-transduced rMSCs on T-cell proliferation was also examined. Results: Rat MSCs could be efficiently transduced with both Ad- and RV-vectors. At a multiplicity of infection of

1186 100, transduction efficiencies measured 2 days after gene transfer were 60% for Ad-vectors. However, there was a dramatic reduction in the percentage of EGFP-positive cells over time (25% on day 14). After retroviral gene transfer and positive selection of gene-engineered MSCs with G-418, purity of transduced cells was 90% which was maintained over the whole observation period (4 weeks). The ability of rMSCs to inhibit T-cell proliferation was unaltered by transduction. Preliminary results from differentiation assays indicate that transduced rMSCs maintain their capacity to diffentiate into chondrogenic and adipogenic lineages, to the same extent as non-transduced cells. Conclusions: We have shown that rat MSCs can be efficiently transduced with gene therapy vectors. Moreover they seem to maintain their immunosuppressive properties indicating the suitability of genetic modification of MSCs for future applications. Supported by Science Foundation of Ireland (SFI 07/IN.1/B925) and Health Research Board (RP/2007/60). E-mail: [email protected] P 234 Identification of tissues differentiated from human embryonic stem cells using a non-typical promoter behavior Tamas I. Orban 1; Agota Apati 1; Andrea Nemeth 1; Nora Varga 1; Virag Krizsik 1; Anita Schamberger 1; Gyorgy Varady 1; Eva Karaszi 1; Laszlo Homolya 1; Katalin Nemet 1; Csaba Miskey 2; Zoltan Ivics 2; Zsuzsanna Izsvak 2; Balazs Sarkadi 1 1Semmelweis

University and National Blood Center, Membrane Research Group of the HAS, Budapest, Hungary; 2MaxDelbruck Center for Molecular Medicine, Mobile DNA Group, Berlin, Germany Human embryonic stem (HuES) cells represent a great potential for gene therapy purposes although apart from technical and experimental difficulties, serious ethical concerns must be resolved before they are actually used in clinical studies. One of the major difficulties of HuES-based applications is how to conduct a directed tissue differentiation. Various attempts are used nowadays including antibiotic selection with tissue-specific promoters or applying artificial drug cocktails to obtain the tissue(s) of interest. Here we describe a novel approach to identify tissues differentiated from HuES cells using a non-typical behavior of the CAG promoter. We generated GFP-expressing HuES clones under the expression of different promoters using the Sleeping Beauty non-viral transposon system or the SEW lentiviral system. Neither of the gene delivery methods modified the subsequent differentiation of these HuES clones, and all kinds of tissue types could be identified after a spontaneous differentiation via the embryoid body formation method. In contrast to other promoter sequences, the CAG promoter yielded exceptionally high reporter expression level in cardiomyocytes, and to a lesser extent, in neuronal type cells. The phenomenon was studied extensively by using confocal microscopy and flow cytometry analysis. To provide evidence that this promoter behavior manifests itself in tissuedependent transcriptional profiles, we collected and studied cell populations with different GFP signal intensities by fluorescence activated cell sorting after differentiating the CAGGFP containing HuES clones. Using real-time quantitative PCR, we could confirm that GFP mRNA levels are higher in cell fractions with greater GFP signal intensity. Moreover,

ESGCT 2008 POSTER PRESENTATIONS cardiomyocyte-specific genes are expressed at significantly higher levels in these “high” GFP cells. We are currently investigating which part(s) of this widely used fusion promoter could be responsible for this non-typical behavior. Taken together, our experiments raise the possibility of using selected promoters for differentiated tissue identifications without any invasive drug-based selection method. E-mail: [email protected] P 235 Mesenchymal stem cells over-expressing ephrin-B2: A potential pro-angiogenic stem cell source Garry Duffy 1; Sinead D’Arcy 1; Tabassum Ahsan 2; Robert M. Nerem 2; Timothy O’Brien 1; Frank Barry

1

1National University of Ireland, Galway, Regenerative Medicine Institute, Galway, Ireland; 2Georgia Institute of Technology, Institute of Bioscience and Bioengineering, Atlanta, GA, United States

Restoration of a vascular supply to ischemic tissues is of high clinical relevance and pro-angiogenic therapies aim to reduce morbidity and mortality rates associated with the onset of cardiovascular disease (CVD). Stem cell therapy has entered into the limelight as a high potential pro-angiogenic therapy. Mesenchymal Stem Cells (MSCs) possess inherent pro-angiogenic capabilities and produce a number of cytokines involved in vessel development. Preclinical studies have reported increased angiogenesis after MSC delivery to the heart and similar outcomes have been reported in recent clinical trials. Stem cell mediated neo-vascularisation has been heightened by the modification of these cells to over express angiogenic cytokines including VEGF and PDGF. In this study we aimed to develop a novel combined stem cell and gene therapy approach that further enhances the pro-angiogenic capability of a cell therapy for promoting angiogenesis. We sought to genetically modify MSCs to over-express a versatile molecule, Ephrin-B2, involved in tissue morphogenesis and vascular development to enhance inherent neovascularisation potential. Using nucleofection, Ephrin-B2 was transiently overexpressed on the cell surface of MSCs to reconstitute embryonic signalling and promote neo-vascularisation. MSCs/ Ephrin-B2 adopted an early endothelial phenotype under EC culture conditions. The cells had an increased ability to form tubules, produce VEGF and incorporate into newly formed endothelial cell tubules. Collectively these data suggest that MSCs/Ephrin-B2 could be a novel pro-angiogenic stem cell source that may be a potential new clinical means for therapeutic angiogenesis in ischemic tissues. E-mail: [email protected] P 236 Use of myogenic transcription factors to enhance the myoregenerative capacity of human mesenchymal stem cells Manuel A.F.V. Gonçalves ; Josephine M. Janssen ; Antoine A.F. de Vries Leiden University Medical Center, Department of Molecular Cell Biology, Leiden, Netherlands There is a growing interest in the use of genetically modified autologous non-muscle cells for treating muscular dys-

ESGCT 2008 POSTER PRESENTATIONS trophies. Human mesenchymal stem cells (hMSCs) may be particularly useful for this goal as these cells (i) are easily obtained from bone marrow or adipose tissue, (ii) can be greatly expanded ex vivo using relatively simple cell culture systems (iii) are efficiently transduced by different types of viral vectors and (iv) can differentiate into many specialized mesodermal cell types including (cardio)myocytes. We previously demonstrated that hMSCs could be transduced with a recombinant full-length Duchenne muscular dystrophy (DMD) gene and subsequently be used to genetically complement dystrophin-deficient skeletal muscle cells by heterotypic cell fusion. However, the efficiency with which the hMSCs engaged in heterotypic cell fusion was low. Accordingly, in this study we tested whether the myogenic fusion capacity of hMSCs could be increased by transducing them with self-inactivating lentivirus vectors encoding naturally occurring and engineered myogenic transcription factors (MTFs). Immunofluorescence microscopy and flow cytometry showed that these MTFs differ in their ability to induce skeletal muscle gene expression in hMSCs. Coupling of the DNA binding domain of human myogenic differentiation factor 1 (hMyoD) to the transactivation domain of the long cardiomyocyte-enriched isoform of human myocardin resulted in a chimeric transcription factor with enhanced myoinductive properties as compared to wild-type hMyoD. Moreover, cell fusion assays performed in co-cultures of DMD myoblasts and hMSCs that did or did not ectopically express a recombinant MTF gene revealed that the activation in hMSCs of a skeletal muscle-specific gene expression program greatly increased their myogenic fusion capacity. On the basis of these experiments, we propose to set up an animal experiment in immunodeficient mice to investigate whether transient induction of MRF activity in hMSCs could increase their contribution to the repair of skeletal muscle damage. E-mail: [email protected] P 237 Next generation mesenchymal stem cells for regenerative medicine applications: antibodies, peptides and lipoplexes for isolation, expansion and genetic modification of mscs Udo Greiser ; Melissa Hoare ; Tyrone Bowes ; Roisin Dwyer ; Daniel O’Toole ; Kaveh Mashayekhi ; Mary Murphy ; Timothy O’Brien ; Frank Barry Regenerative Medicine Institute, National University of Ireland, Galway, Galway, Ireland Background: Successful clinical applications of MSC based cellular therapy will depend on the availability of the most effective cells for the treatment of a specific disease and on large scale production to serve the unmet, growing demand for novel therapeutics of cardiovascular disease and osteoarthritis. Genetic modification by safe, liposome-based vectors may enhance the therapeutic potential of MSCs. Aim: We aim at developing the next generation of MSCs based cellular therapeutics - the most effective cells pulled down from bone marrow (BM) by proprietary MSC-specific antibodies and genetically modified by targeted, peptide enhanced-lipoplexes. Results: We report the isolation of MSC-specific single chain antibodies (scFvs), named TMSC1-4, the successful scFv-assisted pull down and clustering of cells from BM and the successful expansion of these cells. ScFv-assisted magnetic beads sorting of BM has been performed, the target cells have been cultured and passaged up to five times to result

1187 in several T175 cm2 flasks of cells with fibroblast-like morphology. TMSC1-4 showed species cross-reactivity allowing for a rapid and smooth advance of scFvs or scFv-based products like immunoliposomes from in vitro experiments over testing in pre-clinical animal models to human clinical trials. Lipofection of MSCs with reporter genes was enhanced up to ten-fold by including peptides containing reiterated nuclear localization sequences in the liposomal formulation. Transfection with CXCR4 resulted in increased migration towards SDF-1. Peptide-enhanced lipofection, cryo-preservation or scFv-assisted pull-down did not adversely affect the ability of the MSCs to differentiate into adipogenic-, osteogenic- or chondrogenic lineages or their viability. E-mail: [email protected] P 238 Adoptive immunotherapy with EBV-specific cytotoxic CD4 T lymphocytes: new perspectives from a preclinical mouse model of PTLD Anna Merlo 1; Riccardo Turrini 1; Sara Bobisse 1; Rita Alaggio 2; Riccardo Dolcetti 3; Antonio Rosato 1 1University

of Padova, Dept. Oncology and Surgical Sciences, Padova, Italy; 2University of Padova, Dept. Pathology, Padova, Italy; 3CRO, National Cancer Institute, Biotherapy, Aviano, Italy EBV is a human -herpesvirus that latently infects and potentially transforms B cells. In immunocompetent host, the virus-driven proliferation is under the control of a strong cellular immune response directed to latent antigens. When such response fails, such as in transplant recipients, infected B cells can indefinitely proliferate out of control, leading to the development of lymphoproliferative disorders (PTLD). The ethiopathogenesis of PTLD provided the rationale for adoptive immunotherapy approaches with EBV-specific CD8 cytotoxic T lymphocytes (CTL), largely and successfully employed in the prophylaxis and treatment of these malignancies. In this context, a potential direct role of CD4 T cells in controlling B cell proliferation has been poorly investigated. We report that EBV-specific CD4 CTL can be easily generated in vitro from different donors, have a strong proliferative potential and display a lytic machinery fully overlapping that found in CD8 CTL. In a mouse model of PTLD, however, the therapeutic capacity of in vivo adoptively transferred CD4 CTL is partially compromised by down-regulation of MHC-II molecule expression in EBVtransformed lymphoblastoid cell lines (LCL). This phenomenon, observed after LCL transfer in SCID mice but not in human tumors, can be reverted by the use of demethylating agents, such as decitabine, thus restoring the recognition capacity of CD4 CTL in vivo. Therefore, CD4 CTL can be regarded as a potential new tool for adoptive immunotherapy against EBV-related malignancies. E-mail: [email protected]

1188

ESGCT 2008 POSTER PRESENTATIONS

P 239

crease in the developement of ascites in tumor bearing mice. By fluorescent stereomicroscopy, Hospicells were found associated with ovarian adenocarcinoma cells within tumour mass. Tumour obtained by co-injection of both cellular types presented an increase microvascularisation. We have shown an increase of HIF1a expression associated with the presence of the Hospicells and an increase of the synthesis of VEGFA, IL-6 and IL-8 (cell line dependant). Collectively, these data suggest a role of Hospicells in angiogenesis.

Baculovirus vector for mesenchymal stem cells engineering and calvarial bone repair Ching-Kuang Chuang 1; Tzu-Chen Yen 2; Shu-Ju Tu 3; Chin-Yu Lin 4; Kun-Ju Lin 2; Huang-Chi Chen 4; WeiChao Huang 2; Yu-Chen Hu 4 1Department

of Chemical Engineering, Chemical Engineering, Hsinchu, Taiwan; 2Chang Gung Memorial Hospital, Departments of Nuclear Medicine and Neurology, Taoyuan, Taiwan; 3Chang Gung University, Department of Medical Technology, Taoyuan, Taiwan; 4National Tsing Hua University, Department of Chemical Engineering, Hsinchu, Taiwan

E-mail: [email protected]

P 241 Baculovirus has emerged as a promising gene delivery vector. Hereby we demonstrated that baculovirus conferred efficient gene delivery and mediated expression of BMP-2 to therapeutic levels in human mesenchymal stem cells (MSCs). The baculovirus-transduced MSCs enabled osteogenesis in vitro and ectopic bone formation in the back of nude mice, thus implicating the potential of baculovirus-transduced MSCs for gene therapy and bone tissue engineering. Therefore we further implanted the baculovirus-engineered MSCs into rat calvarial bone defects to repair critical-size defects. After surgery, the cranial bone repair was assessed at different time points by X-ray, mineralization quantification, histochemical staining and human nuclear antibody (NUMA) analysis. The repair process was also monitored by molecular imaging (CT and SPECT) to compare the difference in bone density and osteoblastic metabolic activity between the experimental and control groups. These data demonstrated significant calvarial bone repair as a result of baculovirus-engineered MSCs and proved the potential of baculovirus in the context of bone tissue engineering. E-mail: [email protected] P 240 Ascite-derived stromal cells (Hospicells) promote tumorigenicity and angiogenesis Marlene Pasquet 1; Muriel Golzio 2; Eliane Mery 1; Arash Rafii 3; Isabelle Hennebelle 1; Philippe Bourin 4; Justin Teissie 2; Roland Bugat 1; Massoud Mirshahi 5; Bettina Couderc 1 1Institut Claudius Regaud, EA3035, Toulouse, France; 2CNRS, UMR5089, Toulouse, France; 3Institut Claudius Regaud, U563, Toulouse, France; 4Etablissement francais du sang, EFS, Toulouse, France; 5INSERM, UMR736, Paris, France

The microenvironement is known to play a dominant role in cancer progression. Rafii and al. isolated from ascites of patients with ovarian cancer, cells associated with tumoral cells, named Hospicells. Even if these cells do not present specific markers from a known cell lineage, they share some homologies with bone marrow-derived human mesenchymal stem cells and to adipose tissue-derived stromal cells (CD29, CD146, CD166). We studied the role of these cells in ovarian carcinoma progression. In vitro, these cells had no effect on the growth of three human ovarian carcinoma cell lines: OVCAR-3, SKOV1 and IGROV-1. They were chemoattractive for the tumor cells but could also migrate through the tumor cells. In vivo, their co-injection with adenocarcinoma cells enhanced tumor growth whatever the tumor model used (subcutaneous and intra-peritoneally established xenografts in athymic mice). In addition, their injection induced an in-

Long-term effects of cytokine treatment on behavioural recovery and neuronal regeneration in soman-poisoned mice Jean-Marc Collombet 1; Stéphanie Coubard 2; Daniel Beracochea 3; Pierre Filliat 1; Christophe Pierard 2; Guy Lallement 1 1CRSSA,

Département de Toxicologie, La Tronche, France; Département de Physiologie Intégrée, Bretigny Sur Orge, France; 3Université de Bordeaux, UMR CNRS 5228, Talence, France 2IMASSA,

Background: Soman, an organophosphate neurotoxicant, induces long-term neuropathology characterized by a severe neuronal cell death in various brain areas including the hippocampus and amygdala. In the present study, the effects of a cytokine treatment on the neuronal regeneration and behavioural recovery were evaluated. Methods: Subcutaneous injection of a cocktail of 40 g/kg epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) was administered daily to soman-poisoned mice (110 g/kg soman), from post-soman days 1 to 8. Thirty and 90 days after soman intoxication, we investigated the long-term effects of cytokine treatment on emotional reactivity or mnemonic cognitive processes in mice (n  12-20), using fear conditioning tasks or Morris water maze tests, respectively. Mnemonic cognitive processes are known to be supported by hippocampal brain areas while emotional reactivity involves amygdala function. In addition, some mice (n  8-12) were sacrificed on post-soman day 9, 34 or 90 and brains were collected for immunohistochemistry study. Healthy fully-differentiated neurones were quantified on brain sections using neuronal nuclei antigen (NeuN) immunohistochemistry. Results: A clear neuronal regeneration was evidenced overtime in both the amygdala and hippocampus of somanpoisoned mice. The cytokine treatment increased by 15% the neuronal regeneration detected in the hippocampus but did not have any effect in the amygdala. However, the cytokine treatment did not improve hippocampal-driven Morris water maze performances of soman-poisoned mice. Unexpectedly, auditory and contextual conditioned tasks (amygdaladriven emotional reactivity) evidenced behavioural recovery overtime in soman-intoxicated mice and this recovery was accelerated by cytokine treatment. Conclusion: In soman-poisoned mice, cytokine treatment facilitates behavioural recovery depending on amygdala function but this is not related to neuronal regeneration. To the opposite, cytokine treatment increased the neuronal regeneration in the hippocampus but it fails to have positive effects on mnemonic cognitive performances. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS

P 243 Comparison of various promoters driving GFP expression in HEK and human embryonic stem cells, by using lentiviral transduction Dora Turk 1; Agota Apati 2; Gyorgy Varady 3; Tamas Orban 3; Virag Krizik 3; Ferenc Uher 4; Balazs Sarkadi 2; Katalin Nemet 1

duction system. Human embryonic kidney (HEK-293) cells, or undifferentiated human embryonic stem cells (HUES-9), were transduced by the recombinant lentiviruses at a low virus number (MOI). Integrated vector copy number was determined by Q-PCR, while GFP fluorescence intensity was measured by flow cytometry. In HEK cells, containing similar integrated vector copy numbers (1.9  0.6), the SFFV and the CAG promoters resulted in the highest median GFP fluorescence (2548 vs. 1197), while GFP expression driven by PGK promoter was very low (216). In contrast, in the human embryonic stem cells, transduced with the same virus load, similar GFP fluorescence intensities (median  106-337) were observed in the presence of all these different promoter systems. In the currently ongoing experiments, after singlecell cloning of the transduced ES cells, GFP expression is followed during mesenchymal and cardiac differentiation.

P 244

W IT HD RA W N

E-mail: [email protected]

AB ST RA CT

AB ST RA CT

W IT HD RA W N

P 242

1189

1National Blood Transfusion Service, Department of Experimental Gene Therapy, Budapest, Hungary; 2National Blood Transfusion Service, Department of Molecular Cell Biology, Budapest, Hungary; 3Hungarian Academy of Sciences, Membrane Research Group, Budapest, Hungary; 4National Blood Transfusion Service, Experimental Bone Marrow Transplantation, Budapest, Hungary

Human embryonic stem (hES) cells provide an important tool for the analysis of cellular processes and drug development in vitro, and offer a therapeutic potential in a number of different disorders. Genetic modification of hES cells, having a self-renewing capacity, may further extend their field of application. In order to exploit this potential, it is critical to optimize gene transfer and stable gene expression in hES cells, as well as to follow the differentiation of the gene modified cells into various cell types. In the present experiments four different lentiviral vectors were applied, containing either the human cellular polypeptide chain elongation factor1 alpha (EF1) promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), the human phosphoglycerate kinase (PGK) promoter, or the strong enhancer-promoter of spleen focus-forming virus (SFFV). In all cases these promoters were driving GFP expression in a lentiviral trans-

P 245 Successful low-risk hematopoietic cell therapy in a mouse model of type 1 Gaucher disease Ida Berglin Enquist 1; Eva Nilsson 1; Jan-Eric Månsson 2; Mats Ehinger 3; Johan Richter 1; Stefan Karlsson 1 1Laboratory Medicine, Molecular Medicine and Gene Therapy, Lund, Sweden; 2Neuroscience and Physiology, Psychiatry and Neurochemistry, Mölndal, Sweden; 3Lund University Hospital, Pathology, Lund, Sweden

Background: Hematopoietic stem cell (HSC) based gene therapy offers the possibility of permanent correction for genetic disorders of the hematopoietic system. However, opti-

1190 mization of present protocols is required before gene therapy can be safely applied as general treatment of genetic diseases. Methods and results: We have used a mouse model of type 1 Gaucher disease (GD) to demonstrate the feasibility of a lowrisk conditioning regimen instead of standard radiation, which is associated with severe adverse effects. We first wanted to establish what level of engraftment and glucosylceramidase (GCase) activity is required to correct the pathology of type 1 GD mice. We therefore transplanted different ratios of WT bone marrow (BM) cells into lethally irradiated GD mice. Five months after transplantation, a median WT cell engraftment of 2% within the primitive Lin-Sca1c-kit (LSK) compartment corresponding to GCase activity levels above 10 nmol/hr and mg protein was sufficient to reverse pathology within BM and spleen in GD mice with manifest disease. We further applied two different non-myeloablative doses of busulphan (25 and 45 mg/kg) as a pretransplant conditioning regimen. The higher dose allowed robust WT cell engraftment in the range of 2278% in peripheral blood 20 weeks after transplantation. In concordance administration of busulphan at 25 mg/kg with a WT cell engraftment in the range of 1-10% could confer a beneficial therapeutical outcome in this disease model. Moreover, even WT cell engraftment below the detection level within this group, exhibited a significant reduction in glucosylceramide (GlcCer) accumulation and elimination or reduction of disease characteristic Gaucher cells in the spleen and BM. Conclusion: Our data provide encouraging evidence for the possibility to develop safe and efficient conditioning protocols for diseases that only require a low level of normal or gene corrected cells for a permanent and beneficial therapeutic outcome. E-mail: [email protected] P 246 Viral vector mediated delivery of antioxidant and antiapoptotic proteins to mesenchymal stem cells Lisa McGinley 1; Daniel O’Toole 2; Jill McMahon 3; Padraig Strappe 4; Tim O’Brien 1 1National

University of Ireland, Galway, Regenerative Medicine Institute, Galway, Ireland; 2National University of Ireland, Galway, Lung Biology Group, Department of Anaesthesiology, Galway, Ireland; 3National University of Ireland, Galway, Multiple Sclerosis and Stroke Research Group, NCBES, Galway, Ireland; 4University of Sydney, Mind and Brain Research Institute, Sydney, Australia Cell stress and resultant cell death can lead to loss of tissue and organ function and plays a major part in many diseases. Oxidative stress (OS) is implicated in conditions such as atherosclerosis, cancer, macular degeneration and various neurodegenerative disorders. OS occurs when there is a malfunction in the cell’s ability to neutralise free radicals; this results in an insufficient supply of antioxidants and may result in a net increase in the amount of free radicals present in the local cellular environment. This can lead to irreversible cell damage, which may result in mutagenic effects and tissue injury. We have constructed VSV-G pseudotyped, HIV-1 based lentiviral vectors expressing superoxide dismutases, heatshock proteins and catalase. The vectors were titered using real-time quantitative PCR for the viral GAG gene. Testing of the vectors included RT-PCR, western blot and transgene product activity assays. Currently, we are applying this technology to transduce mesenchymal stem cells so that a combined gene and cell therapy approach can be tested in various in vivo models. Our in vitro data suggests these putative therapeutic proteins have a protective effect in terms of cell survival.

ESGCT 2008 POSTER PRESENTATIONS These vectors may be applicable to long term and stable gene expression in the CNS for intervention in neurodegenerative disease or ex vivo stem cell transduction and implantation to myocardial infarct. In summary, lentiviral mediated expression of antioxidant and anti-apoptotic proteins may prove effective by preventing degeneration and promoting cell survival. E-mail: [email protected] P 247 Insertional mutagenesis by retroviral and lentiviral vectors Sean Knight 1; Marieke Bokhoven 1; Abhinav Gupta 1; Mustafa Ceylan 1; Fang Zhang 2; Michael Antoniou 3; Adrian Thrasher 2; Yasuhiro Takeuchi 1; Mary Collins 1 1University

College London, Division of Infection and Immunity, London, United Kingdom; 2Centre for Immunodeficiency, Molecular Immunology, Inst of Child Health, University College London, London, United Kingdom; 3Nuclear Biology Group, Medical/Molecular Genetics, Kings College London School Medicine-Guy’s Campus, Guy’s Hospital, London, United Kingdom

We have established an in vitro assay to assess insertional mutagenesis by retroviral and lentiviral vectors. Isolation of factor independent mutants from the IL-3 dependent cell line Bcl-15 provides a rapid, reproducible and cost-effective method of determining the safety of vectors. Gamma retroviral vector mutants all express IL-3 mRNA, with vector insertions near genes listed as Common Insertion Sites in the RTCGD; such insertions are not observed at the same frequency in control factor dependent cell clones. The lentiviral vector HV, with intact HIV LTRs, an internal SFFV promoter and a WPRE element, generates factor independent mutants at a similar frequency to retroviral vectors, though by a different mechanism. The HV mutants all have insertions in intron 1 of the Ghr gene (growth hormone receptor gene). Interestingly, the HV mutants express a fusion mRNA transcript, which is initiated from the 5 LTR of the vector and splices from the HIV splice donor site to the 2nd exon of the Ghr gene. Two derivatives of the HV vector have been tested in this assay, one containing a Ubiquitin promoter in place of SFFV and the other with a deletion of the WPRE element (WPRE). The HV and WPRE vectors had similar rates of mutagenesis, associated with up regulation of Ghr mRNA; however the Ubiquitin promoter vector produced no mutants. In addition, two SIN lentiviral vectors have recently been tested, the candidate clinical vector UCOE-C and another SIN vector containing the SFFV promoter in place of UCOE. Results with these vectors will be discussed. E-mail: [email protected] P 248 Efficient transduction of human CD34 endothelial progenitor cells using adeno-associated virus vectors Natascha Schuhmann 1; Ombretta Pozzoli 2; Anke Huber 1; Daniele Avitabile 3; Luca Perabo 1; Maurizio Capogrossi 3; Michael Hallek 4; Maurizio Pesce 2; Hildegard Büning 4 1University

of Cologne, Clinic I for Internal Medicine, Cologne, Germany; 2Centro Cardiologico Monzino - IRCCS, Laboratorio di Biologia Vascolare e Terapia Genica, Milan, Italy; 3Istituto Dermopatico dell Immacolata - IRCCS, Laboratorio di Patologia

ESGCT 2008 POSTER PRESENTATIONS Vascolare, Rome, Italy; 4University of Cologne, Clinic I for Internal Medicine and CMMC, Cologne, Germany CD34 endothelial progenitor cells (EPC) possess the unique property to home to ischemic regions where they induce formation of new blood vessels. They can be isolated from cord blood or bone marrow, expanded ex vivo and reapplied efficiently into patients. Efficiency of re-vascularisation can be enhanced by ex vivo gene transfer of pro-angiogenic factors such as vascular endothelial growth factor, angiopoietin-1 or stromal-derived factor 1. Recombinant adeno-associated viral vectors (rAAV) have gained increasing attention as gene transfer system, but their applicability to genetically modify human CD34 cells and, more in general, hematopoietic stem cells is debated. Therefore, we performed a thorough analysis of the interaction between AAV and human CD34 EPC. We compared different human AAV serotypes, assessed the influence of the vector genome conformation on transduction efficiency and performed receptor studies. Furthermore, we correlated the availability of AAV internalisation receptors to the applied culture conditions. Thereby, we identified limiting steps in AAV-mediated gene transfer into human EPC and developed a protocol that allows achieving transduction efficiencies of 57.2  2.6 % in human CD34 cells isolated from cord blood. This high efficiency was further enhanced to 85.6  0.3 % by the addition of retinoic acid and Trichostatin A revealing the impact of transcriptionmodulating drugs on transduction efficiency. These results clearly demonstrate the potential of rAAV as vector system for gene transfer into human CD34 cells. E-mail: [email protected] P 249 Cell infection by capsid engineered AAV vectors: variations from the common scheme? Silke Uhrig 1; Luca Perabo 1; Michael Hallek 2; Hildegard Büning 2 1University

of Cologne, Clinic I for Internal Medicine, Cologne, Germany; 2University of Cologne, Clinic I for Internal Medicine and CMMC, Cologne, Germany The possibility to modify the tropism of adeno-associated virus (AAV) by genetic manipulation of the viral capsid (AAV targeting) has created the possibility for using this virus for a cell or tissue specific gene transfer after systemic application. In addition, targeting vectors are used to perform gene transfer into AAV non-permissive cellss. Since the inserted ligand mediates the cell entry of targeting vectors, it is likely that targeting vectors and AAV with unmodified capsid (wtAAV) differ in their cell entry pathway and intracellular fate. As yet, however, detailed information on the infection biology of targeting vectors is missing. By AAV peptide display selections performed on K562 cells we identified two AAV serotype 2 based targeting vectors, rAAV-B1 and rAAV-C2, that differ by one single amino acid. This variation resulted in a very different phenotype. While rAAV-B1 is able to bind heparan sulfate proteoglycan (AAV2 primary receptor), rAAV-C2 is not. Thus, this pair of targeting vectors is an interesting tool to compare the infection biology of wtAAV and targeting mutants, since rAAVB1 uses a different secondary receptor but the same primary receptor as wtAAV, whereas rAAV-C2 uses different primary and secondary receptors. As hypothesized the new ligand receptor interaction resulted in variations in the intracellular processing in comparison to wtAAV. rAAV-B1 was

1191 e.g. significantly less susceptible to nocodazole- and cytochalasin B-mediated disruption of the cytoskeleton than wtAAV. Furthermore, its transduction efficiency was significantly enhanced by addition of the proteasome inhibitor MG-132, while this drug did not affect wtAAV transduction. Interestingly, variations in the intracellular processing became obvious not only when comparing targeting mutants with wtAAV, but also when comparing the targeting mutants among each other. These, and further results obtained by imaging studies using fluorescent protein-tagged variants of the mutants will be presented. E-mail: [email protected] P 250 Adenovirus type 5 (Ad5) is sequestered and inactivated by binding to coxsackievirus and adenovirus receptor (CAR) and complement receptor 1 (CR1) on human erythrocytes Robert Carlisle 1; Ying Di 1; Anna Cerny 2; Andreas Sonnen 3; Robert Sim 4; Nicola Green 5; Vladimir Subr 6; Karel Ulbrich 6; Robert Gilbert 7; Kerry Fisher 8; Robert Finberg 2; Leonard Seymour 1 1Oxford

University, Clinical Pharmacology, Oxford, United Kingdom; 2Mass Medical School, Division of Medicine, Worcester, United States; 3Oxford University, Division of Structural Biology, Oxford, United Kingdom; 4Oxford University, MRC IHC Unit, Oxford, United Kingdom; 5Hybrid Systems, Vectorolgy, Oxford, United Kingdom; 6Czech Academy of Sciences, Institute of Macromolecular Chemistry, Prague, Czech Republic; 7Oxford University, Division of Structural Biology, Oxford, United Kingdom; 8Oxford University, Clinical Pharmacology, Oxford, United Kingdom Background: Intravenously administered (iv) viral medicines will encounter a variety of innate and adaptive immune components that defend the body against infection. For example human erythrocytes present CAR and CR1. Murine erythrocytes do not. This has critical repercussions for the clinical iv use of Ad5 and also the relevance of murine models for testing Ad5 systemically. Methods: Quantitative PCR was used to assess the binding of Ad5 to erythrocytes from; humans, wild type (WT) mice and transgenic mice presenting CAR or CR1 on their erythrocytes. Circulation kinetics and hepatic infection were also studied in these mice. Results: Ad5 binds to human erythrocytes in PBS ( 90% of dose), binding which is inhibited by the addition of excess fibre protein. There is no such binding to murine erythrocytes. CryoEM shows attachment of Ad5 to human erythrocytes in PBS via its fibre protein, leading to a closer secondary binding. In vitro, ablation of both CAR and factor X mediated Ad5 infection is evident in the presence of human erythrocytes. Furthermore, human erythrocytes in NOD-SCID mice, drastically extend the circulation half-life of iv Ad5. However, despite such an increase, extravasation of the Ad5 into HT29 xenograft tumours is reduced 200x. Almost exclusive binding ( 90%) of Ad5 to human erythrocytes is also observed in plasma. Such binding involves complement, antibodies and CR1. The blood circulation and hepatic infection of Ad5 in WT mice was compared to that in CAR or CR1 transgenic mice. In CAR transgenics circulation is drastically extended, (70% of Ad5 in blood at 6hrs), giving an 800x decrease in hepatic infection. In CR1 mice clearance is equal to that seen in WT mice ( 95%, 10 min), but hepatic infection declines 15x. Polymer coating Ad5 ablates erythrocyte binding in PBS and plasma.

1192 Conclusion: These results show that CAR and CR1 can sequester and inhibit Ad5. Erythrocyte binding will therefore restrict the activity of Ad5 vectors in humans, independent of neutralising antibody status, presenting a formidable barrier to their iv administration. These events are overlooked when testing is performed using conventional murine models. Stealthing of Ad5 using polymers may enable evasion of these natural Ad5 traps. E-mail: [email protected] P 251 Delivery of anti-angiogenic factors into tumors by incorporation of a targeting peptide into adenovirus capsid Betsy Jullienne 1; Frédéric Vigant 1; Erika Muth 1; Ronan Chaligné 2; Céline Bouquet 3; Stéphane Giraudier 2; Michel Perricaudet 1; Karim Benihoud 1 1CNRS,

Univ. Paris-Sud, Institut Gustave Roussy, UMR 8121, Villejuif, France; 2INSERM, U790, Villejuif, France; 3Bioalliance Pharma, Paris, France

Background: Inhibition of new vessels formation constitutes a valuable approach to prevent tumors growth. This can be achieved through adenovirus (Ad)-mediated delivery of anti-angiogenic molecules into endothelial cells. However, their weak expression of the primary Ad receptor, CAR (Coxsackievirus and Adenovirus Receptor), leads to their poor transduction by adenovectors. Methods: To provide Ad with a new entry pathway into neo-vessels, an NGR-containing targeting peptide was inserted into either fiber (AdFNGR) or hexon (AdHNGR) capsid proteins. Results: This strategy provided Ad with a very efficient entry pathway not only in endothelial cells but also in tumoral cells, with the most higher efficacy observed for AdHNGR. Using pharmacological, biochemical and genetic approaches, we demonstrated that AdHNGR was able to bind to CD13 receptors but also to v3 integrins. AdFNGR and AdHNGR were efficient tools to deliver angiostatin K1-5 cDNA into endothelial cells leading to dramatic inhibition of endothelial cells growth through both inhibition of proliferation and cell death induction. Direct administration of AdHNGR into Lewis lung carcinoma (LLC) pre-established in nude mice led to a level of gene transfer comparable to a control Ad. However, pseudotyping AdHNGR with an Ad3 fiber revealed NGR peptide ability to provide Ad with a new entry pathway leading to efficient gene delivery into tumors. As a result, delivery of angiostatin K1-5 cDNA into LLC tumors translated into a stronger inhibition of tumor growth. Conclusion: Altogether, our results pointed out that NGRbearing Ad could constitute valuable tools for treatment of tumors. E-mail: [email protected] P 252 Capsid modified adeno-associated virus vectors identified by directed evolution escape neutralization by human antibodies Stephan Maersch ; Anke Huber ; Michael Hallek ; Hildegard Buening ; Luca Perabo University of Cologne, Internal Medicine, Cologne, Germany

ESGCT 2008 POSTER PRESENTATIONS Background: Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Methods: Capsid libraries were generated by codon randomization of several selected immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Results: Besides testing combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position, maximizing stealth properties and minimizing loss of packaging ability, particle stability and transduction efficacy. We isolated for the first time several capsid variants that remain 100% infective at serum concentrations that completely neutralize AAV2 vectors with wild type capsid. Conclusion: These mutants could be useful for treatment of patients with pre-existing immunity to AAV2 or for therapeutic protocols requiring repeated vector applications. E-mail: [email protected] P 253 Quantitative characterisation of the protein composition of lentiviral vectors by 2D gel approach Jérôme Denard 1; Stéphanie Rundwasser 1; Nicolas Laroudie 2; Florence Gonnet 3; Luigi Naldini 4; Marina Radrizzani 5; Anne Galy 6; Otto-Wilhelm Merten 2; Olivier Danos 7; Fedor Svinartchouk 1 1Genethon,

Service Analyse de Protéines, EVRY, France; RTD, EVRY, France; 3Université d’Evry Val d’Essonne, UMR 8587, EVRY, France; 4San Raffaele Telethon Institute for Gene Therapy, Institut pour thérapie génique, Milan, Italy; 5Molmed SpA, Molmed SpA, Milano, Italy; 6Genethon, REX, EVRY, France; 7Hôpital Necker – Enfants Malades, INSERM U781, Paris, France 2Genethon,

Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. It is known that the parental HIV-1 virus is able to acquire various cellular proteins, which can affect the viral cycle. Indeed, a large number of cellular proteins have been found in preparations of HIV-1 based LV but whether or not they are just co-purified with the vector particles has not yet been determined. We have analysed VSVg-pseudotyped HIV-1 based LV by 2D gel electrophoresis followed by MALDI-ToF. Several types of vector preparations were analysed including those that were extensively purified in the perspective of clinical gene therapy studies. A Proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). This approach allowed identification of proteins present at

ESGCT 2008 POSTER PRESENTATIONS a level of four or more copies per vector particle. We found about 30 C-proteins and only 18 I-proteins associated with LV. These core I-proteins varied in copy number from 5 (AIP1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins were found for the first time in this study. This study defines for the first time, the protein stoichiometry of infectious HIV1-derived lentiviral vector particles. E-mail: [email protected] P 254 Fluorescent HIV-1 integrase fusion proteins for visualization of intracellular preintegration complexes and vector production Vesa Turkki ; Diana Schenkwein ; Hanna Lesch ; HannaRiikka Kärkkäinen ; Anssi Mähönen ; Kari Airenne ; Seppo Ylä-Herttuala University of Kuopio, AIV-institute, Department of Biotechnology and Molecular Medicine, Kuopio, Finland Although lentiviral vectors have emerged as a promising tool for future gene therapy, much is still unknown about the intracellular behaviour of their preintegration complexes (PICs) and their cell-cycle independent transport into the nucleus. To track the intracellular movement of integrase proteins with fluorescence microscopy in vector producing- and transduced cells, we have constructed an integrase (IN) fusion protein by fusing a monomeric red fluorescent protein mCherry to the C-terminus of HIV-1 IN. Fusion protein was introduced to vector particles with direct fusion strategy. The mCherry was cloned into the vector packaging plasmid to the IN open reading frame, leading to IN-mCherry fusions incorporation in the newly forming vector particles as parts of the large gag-pol polyprotein. Third generation self-inactivating HIV-1 based vectors were produced in 293T cells by standard cotransfection of 4 plasmids, one of which contained the IN-mCherry. The packaging of IN-mCherry to both vector like particles and vectors was verified by its fluorescence and by western blots. The functionality of the fusion protein to catalyze vector integration was verified by cell culture assays using HeLa and 293T cells. Fluorescence of IN-mCherry was detected in single viral particles as well as in intracellular and intranuclear PICs. Procedure described here is thus an effective method for studying vector biology from its production until the integration. E-mail: [email protected] P 255 Validation of a triple reporter lentiviral vector encoding eGFP, firefly luciferase and HSV-thymidine kinase in vitro and in vivo Jaan Toelen 1; Abdelilah Ibrahimi 1; Veerle Reumers 2; Caroline Vandeputte 3; Greetje Vande Velde 2; Veerle Baekelandt 2; Zeger Debyser 1; Rik Gijsbers 1 1KU

Leuven, Molecular and Cellular Medicine, Laboratory for Molecular Virology and Gene Therapy, Leuven, Belgium; 2KU Leuven, Molecular and Cellular Medicine, Laboratory for Neurobiology and Gene Therapy, Leuven, Belgium; 3KU Leuven, Nuclear Medicine, Leuven, Belgium Background: Lentiviral vectors are a popular tool for contemporary researchers. Despite the versatility for use both in vitro and in vivo, its limited packaging capacity can impair the use when one wants to combine a therapeutic and a re-

1193 porter gene. We have constructed a lentiviral vector (LV) encoding eGFP, firefly luciferase (fLuc) and HSV truncated thymidine kinase (HSV-tTK) connected with viral 2A-peptide sequences. This triple reporter vector (LV-3R) expresses all three transgenes in an equivalent ratio and with all the functional capacities. Methods and Results: The LV encoding eGFP, fLuc and HSV-tTK was evaluated in vitro after transduction of 293T cells. eGFP was assessed using FACS analysis, fLuc activity was measured using the luciferase assay and HSV-tTK function was evaluated with gancyclovir toxicity. All three transgenes were functional in this in vitro testing. GL261 mouse glioma cells were transduced with LV-3R and sorted for stringent eGFP expression. The cells were subsequently injected in NMRI mice. Bioluminscent imaging (BLI) was used to quantify tumor growth non-invasively. One cohort received gancyclovir treatment whereas the other cohort did not. After two weeks, the treated cohort showed a marked decrease in BLI signal and a reduction in tumor volume was noted after sacrifice of the animals. Conclusion: These results show the versatility of a LV encoding several transgenes using 2A-peptide sequences both in vitro and in vivo. Reporter genes can as such be combined with therapeutic genes in animal disease models. E-mail: [email protected] P 256 Rescue of viral vector integration by LEDGF/p75 fused to alternative DNA binding domains Rik Gijsbers ; Sofie Vets ; Melissa McNeely ; Jan De Rijck ; Zeger Debyser KU Leuven, Molecular and Cellular Medicine, Laboratory for Molecular Virology and Gene Therapy, Leuven, Belgium Background: Lentiviruses stably introduce their viral genome into the host cell chromosome. The transcriptional coactivator LEDGF/p75 is the dominant cellular co-factor for HIV-1 integrase (IN), tethering the preintegration complex to chromatin; however, the exact orchestration of this process remains unknown. LEDGF/p75 organizes the nuclear accumulation and chromosomal localization of IN via its Nterminal domain containing an ensemble of chromatin binding domains. IN interacts with the IN binding domain (IBD, aa 347-429) at the C-terminal end of LEDGF/p75. Stable overexpression of the IBD or the C-terminal end of LEDGF/p75 (325, aa 325-530) strongly inhibit HIV replication by competition for interaction with IN. Methods: We generated strong LEDGF/p75 knock-down lines using miRNA-based hairpins. These cells were stably backcomplemented with chimeric proteins, where we replaced the N-terminal chromatin binding components of LEDGF/p75 (aa 1-324) with alternative DNA binding domains (AR-DBD, H1, H2B, or hepatoma derived growth factor (HDGF)). In addition, we generated chimeric proteins, by replacing the LEDGF PWWP domain with that of HDGF or Hrp2. Results: Upon knock-down, LEDGF/p75 protein was depleted as shown by Western and Q-PCR ( 93% knock-down for the polyclonal cells and 97% for the most potent monoclonal lines). When judged by immunocytochemistry, LEDGF/p75 was depleted from the nucleus. All backcomplementation cell lines were tested for lentiviral vector transduction and HIV-1 infection (NL4.3 NefEnv-fLuc virus or wt NL4.3). Hooking the AR DBD to the LEDGF/p75 C-terminus could not rescue HIV replication. However, with the other DNA-binding protein fusions both LV transduction

1194 and HIV infection were rescued. Whereas fusion of 325 with full length H1 or H2B resulted in moderate rescue, fusion with full length HDGF or the chimeric PWWP-replacements rescued HIV replication and vector transduction to even wild-type levels. Conclusion: Chimeric LEDGF proteins containing alternative DNA binding domains can be substituted for LEDGF, corroborating the tethering role of LEDGF/p75 in viral vector transduction and HIV replication. Our results facilitate the design of safer vectors for gene therapy by fusing the IBD of LEDGF with sequence specific DNA binding motifs. E-mail: [email protected] P 257 Efficiency of stable transduction with integrationdefective lentiviral vectors is tissue-dependent Janka Matrai ; Liesbeth De Waele ; Ling Ma ; Ermira Samara-Kuko ; Abel Acosta-Sanchez ; Thierry VandenDriessche ; Marinee K. L. Chuah KUL-VIB, Vesalius Research Center, Leuven, Belgium Integration-defective lentiviral vectors (LV) obviate the risks associated with insertional mutagenesis and therefore represent a promising platform technology for clinical gene therapy. Integration-defective LVs have been successfully applied for gene delivery into dividing and non-dividing cells in vitro and into various tissue types in vivo. The objective of the present study is to compare the performance of integrating versus integration-defective LVs in mouse liver and spleen in vivo. LV vectors were constructed that expressed a GFP reporter gene or human coagulation factor IX (FIX). Integration-defective LVs were generated by transient cotransfection of vector and helper constructs encoding the prototypical D64V integrase mutant vs. wild-type integrase. In vitro transduction of 293T cells yielded a similar initial % GFP cells up to 48h posttransduction, which remained stable with the integration-competent vectors but gradually declined to basal levels in the subsequent weeks when the integration-defective LVs were employed. The incorporation of scaffold/matrix attachment regions (S/MAR) did not influence the expression kinetics of the integration-defective LVs. In contrast to brain, retina and muscle, GFP transgene expression levels in liver and spleen were significantly lower using integration-defective vs. integrating LVs, as confirmed by confocal microscopy and FACS analysis 3 months post-injection in adult SCID mice. Similarly, genomic integration of LVs was a prerequisite to obtain sustained therapeutic FIX levels in transduced livers of adult SCID mice. These results were consistent with the reduced stable transduction efficiency with integrationdefective LVs, as determined by real-time qPCR and the lack of 2-LTR viral DNA circular intermediates, confirming that the non-integrated extrachromosomal viral vectors are eliminated. We hereby conclude that the efficiency of stable transduction and gene expression with integration-defective LVs varies depending on the target tissue and that genomic integration is a prerequisite for robust stable transgene expression in liver and spleen. *Authors have contributed equally to this work. E-mail: [email protected] P 258 Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system

ESGCT 2008 POSTER PRESENTATIONS Manuel Gonçalves 1; Maarten Holkers 1; Gijsbert van Nierop 1; Roeland Wieringa 1; Maria-Grazia Pau 2; Antoine de Vries 1 1Leiden

University Medical Center, Molecular Cell Biology, Leiden, Netherlands; 2Crucell Holland B.V., Crucell Holland B.V., Leiden, Netherlands A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant fulllength DMD genes). E-mail: [email protected] P 259 Assessment of integration-defective HIV and EIAV vector in vitro and in vivo

Scott Ellis 1; Liang Fong-Wong 2; Sharifah Iqball 1; Vinay Thoree 1; Kyri Mitrophanous 1; Katie Binley 1 1Oxford

BioMedica plc, Biological Systems Group, Oxford Science Park, Oxford, United Kingdom; 2University of Bristol, Henry Wellcome Laboratories for Integrative Neuros, Bristol, United Kingdom Recently there has been much interest in integrase-defective lentiviruses (IDLs) since they could potentially offer the high transduction efficiency and broad tropism of the integrating virus whilst avoiding the potential problems associated with the non-specific integration of a transgene. The aim of this study was to evaluate integration-defective (ID) equine infectious anaemia virus (EIAV) vectors and compare them to ID HIV-1 vectors.

ESGCT 2008 POSTER PRESENTATIONS We generated HIV-1 and EIAV vectors containing identical GFP expression cassettes driven by the SFFV promoter with either a single integrase mutation (D64V), or triple integrase mutations (EIAV: D64V/D116A/E152A; HIV-1: D64V/D116I/E152G), and compared these to the unmutated HIV-1 and EIAV vectors. Both the unmutated and ID vectors were matched for particle numbers based on number of RNA genomes, however when titrated on dividing HEK293T cells, the ID vectors had reduced integration titres of 200fold compared to unmutated vector. These vectors were then compared for their ability to express GFP in a number of cell types over time in vitro including undifferentiated and differentiated C2C12 (murine, muscle) and PC-12 (rat, neuronal) cell lines. Additionally we have compared the degree of transduction and level of expression from these vectors following in vivo rat intrastriatal administration. We are also planning to compare the ratio of integrated to total vector copies in these tissues using ID-PCR. The results from these experiments will be presented and discussed. E-mail: [email protected] P 260 An engineered tethering factor to redirect lentiviral vector integration Melissa McNeely ; Zeger Debyser ; Rik Gijsbers Katholieke Universiteit Leuven, Molecular Medicine, Leuven, Belgium Background: Lentiviral vectors (LV) are promising vehicles for gene therapy purposes; transduction of non-dividing cells as well as their stably integrating genome give these vectors high potential. However, proviral integration into the cellular genome comes with the serious pitfall of insertional mutagenesis. In our research group, LEDGF/p75 has been identified as a molecular tether in lentiviral integration. LEDGF/p75 strongly associates with IN via the integrase binding domain (IBD) located in the C-terminus. The viral preintegration complex is believed to be tethered to transcriptionally active regions in the genome by LEDGF’s PWWP and DNA binding motifs mapped to the N-terminal region. Stable overexpression of the C-terminal end of LEDGF/p75 (325) showed a severe inhibition of HIV replication by blocking integration. Methods: We have now engineered a tethering factor composed of E2C, an engineered polydactyl zinc finger designed to specifically bind a unique 18 bp sequence in the human genome, an eGFP reporter and the 325 fragment of LEDGF. Results: Recombinant MBP tagged E2C-eGFP-325 shows specific in vitro binding to DNA fragments containing the E2C recognition sequence as compared to DNA lacking this 18 bp sequence. In addition, the fusion protein binds to recombinant HIV IN in pull-down assays. Furthermore, we replaced endogenous LEDGF with E2C-eGFP-325 by stable knock-down of endogenous LEDGF and overexpression of E2C-eGFP-325. In these cells, E2C-eGFP-325 localizes in the nucleus and does not evince chromatin binding in mitotic cells. Rescue of HIV infection and LV transduction was weak in comparison with a rescue with WT LEDGF/p75. Conclusion: We examined the feasibility of an engineered tethering factor to control integration site selection of LV. We show that the DNA-binding fragment of LEDGF can be replaced by a 6-Zn finger artificial DNA binding domain. The artificial tether provides specific binding to its recognition site and still binds HIV IN. We are currently verifying

1195 whether this fusion protein can target LV integration in cellular assays. E-mail: [email protected] P 261 Expanded LTRs remain functional in lentiviral vectors Tomas Bos 1; Elke De Bruyne 1; Carlo Heirman 2; Kris Thielemans 2; Karin Vanderkerken 1 1Vrije

Universiteit Brussel, Hematologie en Immunologie Departement, Laarbeeklaan 103, Brussels, Belgium; 2Vrije Universiteit Brussel, Labo voor Moleculaire en Cellulaire Therapie, Laarbeeklaan 103, Brussels, Belgium Background: Modifications to the LTRs have contributed a lot to the popularity and biosafety of lentiviral vectors. Deletions (SIN-vectors) or additions (shRNA-, LoxP-vectors) of specific sequences have demonstrated that alterations to these LTRs do not inhibit their functionality. Sequences cloned into the 3LTR get duplicated during reverse transcription generating double-copy vectors which produce double amounts of transgene compared to single-copy vectors. Since the added modifications never surpassed the size of the U3-deletion (SIN-vectors), the question whether increased size LTRs are still functional remains unanswered. In this study, we investigated if more drastic (size and extra homology) changes inhibit LTR functionality. Methods: Therefore, we gradually added H1 polymerase III promoter-shRNA cassettes to the 3LTR to increase its size in steps of 300 bp. This way, we generated double-copy vectors with double and triple knockdown cassettes (inserts of respectively 600 bp and 900 bp) with an increased homology within the LTRs compared to single knockdown constructs. Results: Vector productions showed no significant decrease in titer compared to the single constructs. An in vitro growing 5T33 multiple myeloma cell line was transduced with all constructs and did not demonstrate any loss of transduction efficiency or stability with expanded LTR-vectors. A genomic DNA PCR amplifying the LTR region of the provirus showed stable integration of the expanded LTR vector. To test the functionality of the expanded LTR, we quantified the knockdown efficiencies of every shRNA-cassette. Our results indicated that all shRNA-cassettes are functional and generate high knockdown efficiencies on qRT-PCR and Western blot. Conclusion: Besides the conclusion that expanded LTRs are functional, we offer a rapid single cloning strategy to combine shRNA cassettes into one single transferplasmid. Compared to combining single non-LTR approaches, our shRNA-technique reduces vector amounts, limits the use of different reporter genes and assures equal delivery of each shRNA to every cell. E-mail: [email protected] P 262 Liver-mediated factor IX expression by AAV8 vector and pre-existing neutralizing antibody in cynomolgus monkey Hiroaki Mizukami 1; Jun Mimuro 2; Akira Ishiwata 2; Fumiko Ono 3; Hiroya Yagi 1; Masashi Urabe 1; Akihiro Kume 1; Terao Keiji 3; Yasuhiro Yasutomi 3; Yoichi Sakata 2; Keiya Ozawa 1 1Jichi

Medical University, Genetic Therapeutics, Shimotsuke, Japan; 2Jichi Medical University, Cell and Molecular Medicine, Shimotsuke, Japan; 3National Institute of Biomedical Innovation, Tsukuba Primate Research Center, Tsukuba, Japan

1196 Adeno-associated virus (AAV) 8-based vectors are promising especially for liver-directed gene therapy approaches. To estimate the efficacy in humans, we utilized cynomolgus monkey (Macaca fascicularis) as a model. After screening of pre-existing neutralizing antibody (NAb) against AAV8 capsid, AAV8 vector encoding macaque Factor IX with a minimal modification (for detection) and a liver specific promoter was injected into 3 young adult male macaques at a dose of 1  1012 or 1  1013 vg/kg. Plasma concentration of the transgene was detectable, but not recognizable as therapeutic level (less than 0.1% of normal) in all animals. On the other hand, the same vector stock showed excellent transgene expression when injected into C57BL/6 mice portal vein. To better understand these results, potential factors affecting transgene expression were analyzed and the presence of neutralizing antibody against AAV8 capsid was suggested. Therefore, we tried to improve the sensitivity of the assay; Target cells, method of detection, and infection conditions were optimized, which resulted in increased sensitivity by more than ten times. As a result, all 3 macaques were proven to be weakly positive for NAb against AAV8, with  1   4. The forth animal was selected based on the sero-negativity by this improved assay. In this animal, intraportal injection of AAV8 vectors (1  1012 vg/kg) resulted in therapeutic levels of Factor IX (30%). Quantitative results with vector DNA of the liver corresponded well with the transgene level (Table). These results imply the importance of the sensitivity of NAb assay, and even low-grade positivity results in significant impairment in gene transfer efficiency. Moreover, when the results were adjusted based on the vector dose per body weight, there is a difference in the levels of transgene expression by around ten-fold. This difference may be explained by species specificity between mouse and macaque, and further study is necessary to validate this issue. E-mail: [email protected] P 263 Lentiviral vectors as a powerful tool to generate transgenic rodent animal models Bavo Heeman 1; Chris Van den Haute 1; Frea Coun 1; Marly Balcer 1; Rik Gijsbers 2; Zeger Debyser 2; Veerle Baekelandt 1 1KULeuven,

Laboratory for Neurobiology and Gene Therapy, Division of Molecular Medicine, Leuven, Belgium; 2KULeuven, Laboratory for Molecular Virology and Gene Therapy, Division of Molecular Medicine, Leuven, Belgium Background: The ability to introduce and express exogenous genes of interest or to knock down the expression of specific genes in animals has become the method of choice to develop novel (gene) therapeutic strategies and to model human diseases like Parkinson’s disease. One approach to generate transgenic animals is to use HIV1 - derived lentiviral vectors (LV) as gene delivery vehicles because they are able to stably integrate into the genome. This technique is especially useful to create non-murine transgenic animals. To evaluate the feasibility and efficiency of different LVbased methods, we generated transgenic mice and rats carrying the green fluorescent protein (eGFP) gene driven by a ubiquitously expressing CMV promoter. Methods: In this study we compared two techniques to create transgenic rodent animals using lentiviral vectors. First, transgenic mice were generated by transduction of preimplantation embryos at morula stage with high titer

ESGCT 2008 POSTER PRESENTATIONS eGFP lentiviral vectors. 24 hours after transduction, eGFP positive blastocysts were transferred into the uteri of pseudopregnant females. Secondly, transgenic rats were produced via injection of lentiviral vectors into the perivitelline space of one-cell stage oocytes. The next day two-cell stage embryos were transferred into the oviduct of pseudopregnant females. Positive offspring was determined via fluorescence microscopy and via PCR on genomic DNA. Results and Conclusion: Both techniques, morula transduction as well as subzonal LV injection of oocytes resulted in the generation of eGFP transgenic mice and rats, respectively. Depending on the vector titer, 15 to 50% of the offspring expressed the eGFP transgene as determined by genomic PCR. 75% of these positive founders had stable germline transmission. Although the efficiency is comparable for both methods, the litter size and the number of successful transfers was at least four-fold higher using the subzonal injection of LV. In addition, the latter technique can be used to generate higher transgenic animals (rats, monkeys), which is an advantage over morula transduction. E-mail: [email protected] P 264 Capsid modified AAV vectors with increased selectivity for melanoma cells Stephan Maersch ; Hildegard Buening ; Anke Huber ; Michael Hallek ; Luca Perabo University of Cologne, Internal Medicine, Cologne, Germany Background: The individual risk of developing malignant melanoma has increased 15-fold in the last 70 years. To date, approximately 1:5000 persons each year become affected by this disease in Germany. Response rates of current therapeutic protocols (chemotherapy and immunotherapy) are poor and severe side effects are associated with the treatment. A promising therapeutic alternative is the use of viral vectors that can selectively infect malignant cells and deliver therapeutic genes that kill the malignant cells (suicide gene therapy) or genes that express immune-stimulatory proteins in order to allow the host immune system to recognize and eliminate cancer cells. However, gene transfer to melanoma cells by currently available viral vectors is hampered by the lack of selectivity of the vector for the targeted tissue. This drawback decreases the efficiency of gene delivery and raises safety concerns. Methods: We applied an evolutionary technique to select modified viruses based on adeno-associated virus of type 2 (AAV2) that infect melanoma cells with increased selectivity. These viruses can be created by insertion of a targeting peptide on the viral capsid. In this way, peptides that are able to bind to cellular receptors expressed on the membrane of melanoma cells will target the virus to the malignant tissue. Specific peptides can be identified by screening a library of randomized peptides inserted on AAV2 capsids and amplifying clones that bind and infect target cells. Results: We isolated two peptides that genetically inserted on the surface of the adeno-associated virus of type 2 (AAV2) capsid, conferred in vitro up to 4-fold increased selectivity in comparison to wt AAV2 vectors. Conclusion: These mutants shall be used to deliver therapeutic genes in melanoma malignant tissues, increasing efficiency and safety of gene transfer. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 265 Computational identification and validation of a promoter for optimizing disease-regulated IL1Ra gene therapy for arthritis Jeroen Geurts ; Onno Arntz ; Miranda Bennink ; Leo Joosten ; Wim van den Berg ; Fons van de Loo Radboud University Nijmegen Medical Centre, Rheumatology Research & Advanced Therapeutics, Geert Grooteplein 28, Nijmegen, Netherlands Promoters of disease-regulated genes are ideal candidates for transcriptional control of transgene expression resulting in gene therapy that matches the intermittent disease-activity course seen in rheumatoid arthritis. In this study we identified a panel of disease-responsive promoters for tailormade gene therapy by comprehensive motif analysis of gene expression profile clusters from an experimental arthritis model. Gene expression profiling was performed on synovial tissue from mice with chronic collagen-induced arthritis, divided in four advancing stages of disease severity. We calculated 234 differentially expressed genes whose expression was at least tenfold-regulated in at least three severity stages using Expander 4.0 software. Next, the expression profiles were partitioned into eight distinctive clusters using the kmeans algorithm. Since promoters containing a TATA-box between positions 34/ 29 relative to the transcription start site are strongly associated with transcriptional specificity, we identified promoters fulfilling this prerequisite by screening the upstream sequences of the murine genes and human orthologues with two TATA-box position weight matrices using PATSER. The transcription factors putatively governing the eight profiles were elucidated by calculating overrepresented cis-regulatory sequences in the proximal promoters ( 500/200). Immediate-early expression profiles were strongly correlated to the presence of NFB and AP-1 binding sites, while profiles correlating to disease severity were linked to C/EBP/, AP-1, and Pu.1 sites. Next, we addressed the efficacy of disease-regulated interleukin-1 receptor antagonist (IL1Ra) gene therapy, which requires a strong promoter in order to provide the necessary 100-fold excess over IL-1. To this end, we selected the Saa3 promoter ( 314/50), containing a single NFB and three C/EBP sites, as a promising candidate. Using an in vitro system for assaying IL1Ra efficacy based on activation of NFB by IL-1, we managed to demonstrate efficacy comparable to a CMV promoter. These results demonstrate the value of a computational approach for identifying regulated promoters for optimal gene therapeutic treatment of arthritis E-mail: [email protected] P 266 Development of a novel baculovirus vector that enable prolonged transgene expression Wen-Hsin Lo ; Ching-Kuang Chuang ; Hsiao-Chiao Shiah ; Yu-Chen Hu National Tsing Hua University, Department of Chemical Engineering, Hsinchu, Taiwan The baculovirus (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) have been claimed safe and widely employed as an attractive gene therapy vector in mammalian cell system. However, the transient expression nature due to its incapability to replicate in mammalian cells

1197 limits its application. In this study, we aimed at developing novel baculovirus vectors that carry the Epstein-Barr virus orip and EBNA-1, which are essential for the episomal maintenance of the EBV genome in latently infected cells, and transgene expression cassette based on FLP/Frt system. In this system, the FLP recombinase catalyzes highly efficient site-specific recombination, ultimately resulting in the circularization of Frt-flanked DNA and creation of a replicated episomal plasmid to solve the problem of transient expression of baculovirus in the transduced mammalian cell. For the purpose, we constructed a binary baculovirus-mediated episomes delivery system that consists of one baculovirus vector for the expression of FLP recombinase and the other baculovirus vector that contains all of expression cassette flanked by Frt sites. The results showed that episomallymaintained plasmids were rapidly generated at day 2 after co-transduction. The efficiency of episomes delivery remained 4080% in several different cells containing primary cells and mesenchymal stem cells. Sustained transgene expression in Hek293 cells were observed for over 35 days under no selective pressure. In the presence of slightly selective pressure (50 g/ml G418), the transgene expression lasted for 60 days. Furthermore, we genetically engineered hMSCs with the baculovirus-mediated episomes delivery system expressing bone morphogenetic protein-2 (BMP-2). The BMP-2 expression could be enhanced and further accelerated differentiation of hMSCs into osteoblasts. We successfully combined the abilities of FLP/Frt and OriP/EBNA1 to develop an novel baculovirus-based strategy that could mediate prolonged episomal transgene expression and without insertional mutagenesis. This novel baculovirus vector has potential for gene therapy. E-mail: [email protected] P 267 Efficient transduction of adult human and rat mesenchymal stem cells with a third generation HIV-1 derived lentivirus Barry McGrath ; Timothy O’Brien Regenerative Medicine Institute (REMEDI), Medicine, National University of Ireland Galway (NUIG), Galway, Ireland Background: Due to their pluripotency and their ability to differentiate into various different cell types including cartilage, bone, fat and other connective tissue, mesenchymal stem cells (MSCs) offer tremendous potential in regenerating diseased or injured tissue. Lentiviral-transduced MSCs are of interest to gene therapy as they can be used to deliver genes to sites of injury after systemic delivery, serve as a platform for controlled therapeutic gene expression and have the potential for modification by over-expression of differentiation factors. Therapeutic use of transduced MSCs must satisfy questions regarding biosafety issues, such as the effect of transduction on differentiation capability, and the potential risk of insertional mutagenesis associated with integrating vectors. Methodology: A third generation lentiviral vector (pRRLcPPT-PGK-GFP-WPRE), pseudotyped with VSV-G, was used to transduce rat and human MSCs at varying MOI and different passage numbers to assess the effect of viral titre and passage number on MSC transduction efficiency. The ability of lentiviral-transduced MSCs to differentiate into various cell types was assessed using various differentiation assays. The insertion site preference of the lentivirus in hMSCs was assessed using LAM-PCR.

1198 Results: Minor differences in transduction efficiencies were observed at different passage numbers for both rat and human MSCs. A positive dose-response for viral titre on transduction efficiency of both rMSCs and hMSCs was also observed. Transduced hMSCs demonstrated the ability to differentiate into various cell lineages. Preliminary data on the insertion site preferences suggests that transcribed regions of hMSCs are favoured insertion sites with 2/3 of the insertion sites characterised (n  31) being located in/near transcribed regions. This part of the study is ongoing Conclusions: HIV-derived lentivirus can efficiently transduce both rat and human MSCs. No apparent affect on the ability of lentiviral-transduced MSCs to differentiate along various cell lineages was observed. HIV insertion site preferences in hMSCs highlights the potential risk of insertional mutagenesis. E-mail: [email protected] P 268 Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor-binding and limits gene transfer to liver Frédéric Vigant 1; Delphyne Descamps 1; Betsy Jullienne 1; Stéphanie Esselin 1; Elisabeth Connault 1; Paule Opolon 1; Thierry Tordjmann 2; Emmanuelle Vigne 3; Michel Perricaudet 1; Karim Benihoud 1 1CNRS,

Univ. Paris-Sud, Institut Gustave Roussy, UMR 8121, Villejuif, France; 2INSERM, U757, Orsay, France; 3Sanofi-Aventis, , Vitry-sur-Seine, France Background: Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. Results: The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, while HVR5-modified Ad were still able to efficiently transduce different cell lines, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Conclusion: Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile. In particular, Ad5-HVR5 modification could be combined with other capsid-protein modifications made to enhance Ad infectivity in different tissues or to further reduce hepatocytes transduction. E-mail: [email protected] P 269 A modular bidirectional expression system for gammaretroviral and lentiviral vectors Tobias Maetzig 1; Melanie Galla 1; Rainer Loew 2; Christopher Baum 3; Axel Schambach 3 1Hannover Medical School, Experimental Hematology, Hannover, Germany; 2Eufets AG, Eufets AG, Idar-Oberstein,

ESGCT 2008 POSTER PRESENTATIONS Germany; 3Hannover Medical School, Experimental Hematology, Carl-Neuberg-Str.1, Hannover, Germany Background: Efficient gene therapy of polygeneic diseases as well as the simultaneous expression of therapeutic and marker genes in cell and animal studies depends on potent co-expression strategies. To overcome limitations by uneven expression levels (IRES) or protein alterations (2A cleavage), Amendola et al. (NatBiotechnol, 2005) recently published a lentiviral co-expression system based on a bidirectional internal promoter cassette (Baron et al., NAR., 1995) leading to high expression levels of two unaltered transgenes. Aim: We wanted to construct a modular bidirectional expression system based on Murine Leukemia Virus – SIN vectors which are easy to produce in stable packaging cells. In parallel, we constructed lentiviral vectors to analyze the performance of the bidirectional cassettes independent of the integration process. Results: We constructed a bidirectional promoter cassette consisting of an internal promoter sharing its enhancer with an anti-sense orientated minimal CMV (mCMV) promoter and cloned it into gammaretroviral and lentiviral backbones in a modular fashion. Having tested DsRed, tdTomato, tCD34, mCherry and EGFP as transgenes, optimal performance was dependent on their orientation. For genes with potential splice sites, antisense expression was more efficient. This was also exemplified by the intron-containing EF1a promoter, of which only the anti-sense intron was efficiently transduced. Of note, different promoters fused to the mCMV mediated efficient co-expression with different intensities (SFFV PGK EF1a). Besides transferring integration competent lentiviral vectors, we could also transiently deliver bidirectional vectors when packaged with the integrationdefective D64V-integrase mutant. High gene expression could be observed 40hrs after transduction with residual transgene levels still present 3 days later. Conclusion: From a total of 40 vectors, we identified optimal promoter transgene combinations for bidirectional gammaretroviral and lentiviral backbones. The vector series may be of great interest for the delivery of cDNAs along with reporter genes, and for potential clinical applications (e.g. expression of TCRs or antibodies). E-mail: [email protected] P 270 Differential transgene expression profiles from rAAV2/1 vectors using the tetON and CMV promoters in the rat brain Olivier Bockstael 1; Abdelwahed Chtarto 1; Janika Wakkinen 2; Xin Yang 1; Catherine Melas 1; Marc Levivier 1; Jacques Brotchi 1; Liliane Tenenbaum 1 1Université Libre de Bruxelles, Lab. Exp. Neurosurgery, Brussels, Belgium; 2University of Helsinki, Department of Medical Genetics, Helsinki, Finland

The biodistribution of transgene expression in the CNS after localized stereotaxic vector delivery is an important issue for safety of gene therapy for neurological diseases. The cellular specificity of transgene expression from rAAV2/1 vectors using the tetON expression cassette in comparison with the CMV promoter was investigated in the rat nigrostriatal pathway. After intrastriatal injection, although GFP was mainly expressed into neurons with both vectors, the percentage of GFP astrocytes was 5-fold higher with the CMV vector. The relative proportions of DARPP-32 pro-

ESGCT 2008 POSTER PRESENTATIONS jection neurons and parvalbumin interneurons were respectively 13:1 and 2:1 for the CMV and tetON vectors. DARP32 neurons projecting to the globus pallidus were strongly GFP with both vectors, whereas those projecting to the substantia nigra pars reticulata (SNpr) were efficiently labeled by the CMV but poorly by the tetON vector. Numerous GFP cells were evidenced in the subventricular zone with both vectors. However, in the olfactory bulb (OB), GFP neurons were observed with the CMV but not the tetON vector. We conclude that the absence of significant amounts of transgene product in distant regions (SN and OB) constitutes a safety advantage of the AAV2/1-tetON vector for striatal gene therapy. Midbrain injections resulted in selective GFP expression in tyrosine hydroxylase neurons by the tetON vector whereas with the CMV vector, GFP cells covered a widespread area of the midbrain. The biodistribution of GFP protein corresponded to that of the transcripts and not of the viral genomes. We conclude that the rAAV2/1-tetON vector constitutes an interesting tool for specific transgene expression in midbrain dopaminergic neurons. E-mail: [email protected] P 271 Analysis of reverse transcription thermo-sensitivity in lentiviral vectors with different envelope proteins Marlene Carmo 1; João D. Dias 1; Amos Panet 2; Ana S. Coroadinha 1; Manuel J. T. Carrondo 1; Paula M. Alves 1; Pedro E. Cruz 1 1ITQB-UNL/IBET,

Animal Cell Technology Laboratory, Oeiras, Portugal; 2Hebrew University, Hadassah Medical School, Department of Virology, Jerusalem, Israel Background: Lentiviral vectors have emerged as promising tools for gene therapy. They present the advantages of gamma retroviral vectors coupled with the capacity to efficiently transduce non dividing cells. However, like gamma retroviral vectors they display low stability, affecting the production, storage and their transduction efficacy. Our previous results demonstrated that the reverse transcription thermo-sensitivity is responsible for gamma retroviral vectors inactivation. The aim of the present work is to understand whether the reverse transcription process thermo-lability in lentiviral vectors is also responsible for virus inactivation and furthermore whether different envelope proteins–amphotropic and RDpro–affect viral thermo-sensitivity. Methods: Lentiviral vectors, encoding GFP, were produced by STAR-A and STAR-RDpro packaging cell lines transfected with pHRSIN-CSGW. To investigate the mechanism and kinetics of viral thermo-sensitivity, purified vector preparations were incubated at 37°C and tested for infectivity, endogenous reverse transcription activity, DNA polymerase activity and viral RNA degradation. Furthermore, the residual activity of the vector to enter host cells (RNA and p24 capsid protein entry) was also tested. Results/Conclusions: The res ults indicate that, for both amphotropic and RDpro pseudotyped lentiviral vectors, the capacity to perform reverse transcription decreases during incubation at 37°C with a profile very similar to that of the infectivity loss. Interestingly, RT DNA polymerase activity and viral RNA were rather stable at 37°C, not affecting the rate of reverse transcription decline. Moreover, the amount of viral RNA and of p24 capsid protein of the pre-incubated vectors that entered the host cells, upon infection, decreased slowly during incubation at 37°C, and at a similar rate for

1199 both lentiviral pseudotypes. Taken together it was possible to conclude that the thermo-sensitivity of the reverse transcription process is a mechanism of lentiviral vectors inactivation independently of the envelope protein at the surface of the vector. Stabilization studies of the reverse transcription process should be pursued in order to improve the production and transduction of lentiviral vectors. E-mail: [email protected] P 272 Retroviral vector production in serum-free media: the role of serum lipids Ana F. Rodrigues ; Ana I. Amaral ; Paula M. Alves ; Manuel J.T. Carrondo ; Ana S. Coroadinha IBET/ITQB-UNL, Animal Cell Technology Unit, Oeiras, Portugal Background: The manufacturing of retroviral vectors still represents a highly challenging task mainly due to the low productivities of the packaging cell lines, reduced stability of the vector and low production under serum-free conditions. The reduction of serum in the culture media results in a pronounced decrease in viral productivity. The major causes for the limitations in viral production in serum-free media still remain unclear and are the subject of this work. Methods: The effect of reducing the serum concentration in the culture media on the viral production was analyzed in terms of cell growth, metabolic behavior, viral productivities and vector characteristics using two different packaging cell lines: 293 FLEX 18 and Te Fly Ga 18. Results: The reduction of serum in the production media resulted in a decrease in infectious vectors productivity. However, the total particles productivity was maintained, as assessed by measuring the viral RNA and the gag-pol expression. The absence of the serum lipid fraction was found to be the major cause for the decrease in viral productivity under serum-free conditions. The use of delipidated serum confirmed this observation and culture media supplementation with lipids resulted in a 9 fold increment in viral productivity allowing not only the total recovery of viral titers, but also an additional 4 fold increment. The adaptation of the cell lines to reduced-serum conditions showed that Te Fly Ga 18 can adjust and recover viral productivities after 2 weeks whereas 293 FLEX 18 shut off viral production. A characterization of lipid biosynthetic capacity of the cell lines using 13C-NMR Spectroscopy revealed that this adaptation period induced an increase in lipid biosynthetic capacity, particularly cholesterol, for Te Fly Ga 18 but not for 293 FLEX 18. Conclusion: In this work one of the major bottlenecks of retroviral vector production in serum-free conditions was identified. The ability to produce retroviral vectors at higher titers and with high stability in serum-free conditions is dependent upon the cell lipid metabolism, particularly cholesterol, making it a good candidate for manipulation in the culture media. E-mail: [email protected] P 273 Ty3 yeast retrotransposon integrase activity in human cells Nicholas Casey ; Greg Woods University of Tasmania, Menzies Research Institute, Hobart, Australia

1200 Background: Integrating gene therapy vectors, while providing efficient and stable transgene expression, carry a risk of insertional mutagenesis. Consequently, there has been much research into improving their safety. Retrotransposons are related to retroviruses, but lack the machinery for lateral transfer between cells. In the Ty3 yeast retrotransposon, selection pressure for minimal disruption to the host genome has resulted in a targeting mechanism that integrates the retrotransposon immediately upstream of Pol-III class, tRNA genes. The retention of such characteristics in human cells would make this targeting mechanism very attractive for gene therapy purposes. Ty3 integrase associates with human tRNA genes expressed in yeast cells and a chimeric form is active but non-specific in human cells. We tested the activity of the complete or ‘wild type’ integrase in human cells. Methods: We constructed a vector co-expressing the Ty3 Integrase with a growth-selection marker. The same vector, lacking the integrase, acted as a control. We also constructed a marker ‘integrant’ of a Green Fluorescent Protein (GFP) gene flanked by Ty3 retrotransposon Long Terminal Repeats. This integrant and the integrase-expression or control vectors were co-transfected into 293T cells by Calcium Phosphate precipitation transfection. Results: Initial GFP expression was widespread in both groups, indicating successful transfection of the transgenes. As expected, fluorescence was lost from some or all cells of many colonies. This was ascribed to transient expression. Similarly, a certain rate of ‘passive integration’ was expected in the control group, evinced as stable expression in the absence of integrase. Importantly however, where the Ty3 integrase was expressed, the proportion of colonies exhibiting stable, ubiquitous expression of the GFP marker was manyfold higher than in controls. Conclusion: These results indicate that the ‘wild-type’ Ty3 integrase is indeed active in human cells and can result in stable transgene expression. E-mail: [email protected] P 274 Tissue specific expression from integration deficient lentiviral vectors Sonia Ferreira 1; Pascal Leclere 1; Vincent Kao 1; Gillian Talbot 1; Olivia Bales 1; Michael Antoniou 2 1King’s

College London, Medical and Molecular Genetics, London, United Kingdom; 2King’s College London, Medical and Molecular Genetics, London, United Kingdom Lentivirus-derived vectors are among the most promising viral vectors for gene therapy currently available, but their use in clinical practice is potentially limited by the associated risk of insertional mutagenesis. Previous reports have shown that efficient and sustained transgene expression can be achieved in non-mitotic cells such as ocular and muscle tissues with integration defective lentiviral vectors (ID-LV) under the control of ubiquitous viral promoters, namely those of Spleen Focus Forming Virus (SFFV) and Cytomegalovirus (CMV). However, it is still unclear whether genomic tissue-specific promoters can direct expression from an ID-LV context. We have therefore embarked on a comparative investigation to assess the ability of tissue-specific promoter elements [erythroid human b-globin, HBB; muscle human desmin, DES; muscle creatine kinase, CK-M] to express from within ID-LV. Expression cassettes consisting of the these promoters driving a GFP reporter gene have been constructed within a standard SIN LV and viruses generated in both an ID and a standard integrating configuration as a control. LV

ESGCT 2008 POSTER PRESENTATIONS are then functionally analysed in a range of appropriate tissue culture cell lines. Transgene expression is determined by FACS, and LV copy number, status and integrity by Southern blotting and Q-PCR. Our results provide the first evidence that the utility of ID-LV can be extended to provide tissue-specific therapeutic gene expression. E-mail: [email protected] P 275 Chromatographic purification of adenoviral vectors for gene therapy Marc Eglon 1; Aoife Duffy 1; Timothy O’Brien 1; Padraig Strappe 2 1National

University of Ireland, Galway (NUIG), Regenerative Medicine Institute (REMEDI), Orbsen Building, NUIG, Galway, Ireland; 2University of Sydney, Brain and Mind Research Institute, Sydney, Australia Introduction: With the increasing use of adenoviral vectors in vaccine development and gene therapy, there is an escalating focus on the development of quality-focused purification protocols that can be translated to GMP production. HPLC is emerging as the preferred mode of purification, enabling the production of vectors at a clinically relevant scale and quality. This study focuses on the development of a 2-step HPLC process consisting of anion exchange and gel filtration. Virus Production: HEK293 cells were grown in 10-layer CellStacks™ [Corning] and infected at MOI 10 with Ad5-GFP. Cellbound virus was harvested by low-speed centrifugation after 48-72 hours, when 100% CPE was observed. Virions were liberated by serial freeze-thaw cycles and free DNA was digested using benzonase. The crude lysate was clarified by low-speed centrifugation and subsequent filtration. HPLC: Clarified material was purified by anion exchange [Q-Sepharose XL––virus licensed] and gel filtration [Sepharose 4 Fast-flow] using the Akta Explorer 100 Air HPLC system. During process optimization, HPLC fractions were added to HEK 293 cells and transduction monitored after 24 hours by fluorometry to confirm the presence of potent virus. Quality Control: Purified adenovirus was assessed to demonstrate benzonase clearance [ELISA], purity [Silver Stain], virion viability [Plaque Assay] and the absence of replication-competent adenovirus [PCR]. Results: We developed a robust scalable process, capable of producing high-quality adenovirus at a titre that is suitable for scale-up and clinical translation. The process can be performed in only a few hours, uses low-cost buffers and requires minimal operator intervention so is economically and logistically viable for GMP adenovirus production. E-mail: [email protected] P 276 Vpr-independent packaging of functional HIV-1 integrase fusion proteins into third generation lentivirus vectors Diana Schenkwein ; Vesa Turkki ; Hanna-Riikka Kärkkäinen ; Hanna Lesch ; Seppo Ylä-Herttuala University of Kuopio, Department of Biotechnology and Molecular Medicine, Kuopio, Finland Lentivirus vectors are efficient gene transfer tools that integrate transgenes into the genomes of target cells. The in-

ESGCT 2008 POSTER PRESENTATIONS tegration pattern of HIV-1 favors active genes and transcriptionally active genomic areas, which may lead to insertional mutagenesis. To study the possibility of modifiying the integration pattern of HIV-1, different approaches have been used. HIV-1 integrase (IN) fusion proteins as well transposases or integrases from other origins have been combined with HIV-1 vectors to study the feasibility of integration pattern modification. Usually IN-fusion proteins have been incorporated into virions with a so called trans-packaging method that relies on the expression of HIV-1 Vpr. The transpackaging method is not directly applicable to third generation lentivirus vector production, which led us to develop another way of incorporating heterologous proteins into virions. The 3rd generation HIV-1 packaging plasmid was modified to contain different IN-fusion proteins cloned to the 3end of pol. Vectors were produced with standard CaPo4 transfection of 293T producer cells using the modified packaging plasmids. The IN-fusion proteins were found to incorporate into virions, and to retain the ability to catalyze integration. In addition, the tested fusion proteins also had distinct effects on cells upon transduction, depending on the fusion partner used. The direct IN-fusion protein packaging strategy was found to be applicable to packaging both proapoptotic and fluorescent marker proteins into vectors. Third generation HIV-1 vectors can thus be incorporated with different IN-fusion proteins independent of Vpr expression, and functional vectors can be produced without modifications in the production protocols. We expect our packaging method to be useful for different applications, among others those that currently necessitate a double transduction of cells with two different lentivirus vectors. E-mail: [email protected] P 277 Comparative study of rAAV2/7 purification methods for transduction in the mouse brain following serum free production Jaan Toelen 1; Anke Van der Perren 2; Martine Michiels 1; Irina Thiry 1; Chris Van Den Haute 2; Rik Gijsbers 1; Veerle Baekelandt 2; Zeger Debyser 1 1KU

Leuven, Molecular and Cellular Medicine, Laboratory for Molecular Virology and Gene Therapy, Leuven, Belgium; 2KU Leuven, Molecular and Cellular Medicine, Laboratory for Neurobiology and Gene Therapy, Leuven, Belgium Background: Recombinant adeno-associated viral (rAAV) vectors have recently been described to be efficient vehicles for gene transfer into the central nervous system. Previous studies using viral vectors have shown that production and purification methods greatly influence the immunogenicity and toxicity after stereotactic injection in the mouse brain. Methods: Recombinant AAV vectors were produced under serum-free conditions in 293T cells using a CMV-eGFPT2A-Fluc expression cassette and packaged with AAV7 capsid (rAAV2/7). Vectors were further purified and concentrated using ultracentrifugation, an iodyxanol gradient or size-exclusion concentrators. Each vector batch was stereotactically injected undiluted and a 20-fold dilution into the mouse striatum. Transduction efficiency and distribution of the vector was determined by in vivo bioluminescence imaging (BLI), immunohistochemical analysis of eGFP expression and Cavalieri measurements of transduced volume. Locoregional toxicity was assessed using a Chresyl-Violet staining procedure. Results and Conclusion: All purification methods resulted in functional AAV2/7 vector under serum free con-

1201 ditions, showing efficient transduction of the mouse striatum. Dilution of the vector resulted in 20-fold decrease of the BLI signal and a smaller eGFP transduced volume. Colocalisation of eGFP and NeuN demonstrated efficient transduction of neuronal cells. Some undiluted vector preps transduced excessively outside the striatal area and resulted in mild toxicity. After dilution, no toxicity could be detected whereas the striatum was still efficiently transduced. Our initial results underscore the relevance to study new rAAV production and purification methods for gene delivery in the brain. E-mail: [email protected] P 278 Utilization of modified in vitro assembled mason-Pfizer monkey virus particles Pavel Ulbrich ; Irena Vorackova ; Tibor Fuzik ; Tomas Ruml Institute of Chemical Technology, Prague, Dpt. of Biochemistry and Microbiology, Prague 6, Czech Republic An alternative for utilization of genetically engineered viruses in gene therapies is the usage of pseudoviral particles made of viral structural proteins assembled into viruslike particles in vitro. The advantage of this system is a lack of particle infectivity and replication. Purified deletion mutant of Mason-Pfizer monkey virus (M-PMV) structural polyprotein Gag can be used for very efficient assembly of spherical virus-like particles in vitro. Appropriate viral structural proteins were purified in sufficient amounts and purity and the system of particle formation was optimized. We studied the incorporation of various therapeutically utilizable compounds into these particles. We found that different types of nucleic acids and other polyanions can be incorporated into formed particles. Various RNAs were incorporated with higher efficiency in comparison with plasmid DNA. On behalf of improving plasmid DNA incorporation we decided to modify viral structural protein. We have found that N-terminal modification of viral structural protein does not affect particle formation and that the modification is exposed on the particle surface. We selected short oligopeptides that should interact with prostate specific membrane antigen. By immunofluorescent microscopy we tested interactions of our particles with prostate cancer cells (LNCaP). Also Western blot method was used for determination of particle localization and cell entry. The entry of particles with incorporated pGFP DNA into LNCaP cells was also determined by fluorescent microscopy. This work was supported by research grants KJB501270701, KAN208240651, MSM6046137305 and 1M6837805002NR8797. E-mail: [email protected] P 279 Development of a new helper vector for the production of helper-dependent adenovectors using a baculovirus/ adenovirus hybrid system Zaid Aburubaiha 1; Mahmoud Srour 1; Henry Fechner 2; Johannes Oldenburg 1; Rainer Schwaab 1 1University

of Bonn, Inst. f. Exp. HÃmatologie u. Transfusionsmedizin, Bonn, Germany; 2Charite UniversitÃtsmedizin Berlin, Department of Cardiology and Pneumology, Berlin, Germany

1202 The ultimate form of Adenovirus vector modification is the creation of fully deleted Helper-Dependent (HD) Adenovectors which lack all the adenovirus genes except the replication and packaging elements. HD adenovectors elicit the least immune response and can accommodate an insert of up to 36 kb which provide enough space for large genes such as coagulation factor VIII and Dystrophin genes. HD adenovectors are produced in the presence of a helper adenovirus that supplies the viral genes in trans. The use of a helper virus almost always results in contamination of the HD adenovectors with the helper virus which is a major disadvantage in their production. For this reason, we have designed a novel system for the production of HD adenovectors to avoid contaminating helper virus. In this system, a packaging-deficient, replication-competent helper genome is delivered to HEK 293 cells, transfected with a HD adenovector, by a recombinant Baculovirus/ Adenovirus Hybrid. The adenovirus genome lacking the the ITRs and packaging signal but flanked by AAV ITRs was cloned into the baculovirus expression system (Invitrogen). The resulted recombinant helper Baculovirus is used to infect HEK 293 cells which then provides the helper function needed for the production of the HD vector. The results will be presented and discussed. E-mail: [email protected]

ESGCT 2008 POSTER PRESENTATIONS P 280 Comparative analysis of UCOE-based lentiviral vectors in a murine neonatal intravascular delivery system Sonia Ferreira 1; Simon Waddington 2; Michael Antoniou

1

1King’s

College London, Medical and Molecular Genetics, London, United Kingdom; 2University College London, Department of Haematology, London, United Kingdom Ubiquitous chromatin opening elements (UCOEs) consist of dual divergently transcribing promoters from housekeeping gene loci and have been found to be resistant to epigenetic-mediated silencing. Recent work has assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector, reporting high reproducible expression with clear evidence for resistance to silencing. We have therefore embarked on an investigation to assess the reproducibility and stability of expression from the A2UCOE element either alone or linked to the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or the ubiquitous expressing promoter/enhancer from Cytomegalovirus (CMV) from within lentiviral vectors. In vivo testing via mouse neonate (1-2 day old) systemic intravascular injection was conducted and tissue from several organs was collected after a 6-week latency period. Analysis of vector copy number and eGFP reporter gene expression together with immunohistochemical characterisation are being undertaken. Our preliminary data suggest that the A2UCOEWPRE combination provides good levels of expression in the absence of enhancer activity thereby providing a high safety profile in gene therapy applications. E-mail: [email protected]