Ventricle Using the Stiletto⢠Endocardial Direct. Injection Catheter System. Qing Wang, Yawen L. Chiang. 1Preclinical, Corautus Genetics, Inc., San Jose, CA.
CARDIOVASCULAR: MYOCARDINAL GENE THERAPY Conclusion: The same plasmid behaves completely differently in different tissues. Transgene expression efficiency does not directly correlate to plasmid uptake efficiency after naked DNA injection in skeletal and cardiac muscles in rats. This observation questions the relevance of indirectly testing plasmid DNA uptake by monitoring transgene expression.
925. pVGI.1 (VEGF2) Biodistribution in Swine Following Injection into Anterior Wall of the Left Ventricle Using the Stiletto™ Endocardial Direct Injection Catheter System Qing Wang, Yawen L. Chiang. 1 Preclinical, Corautus Genetics, Inc., San Jose, CA. Intramyocardial delivery of vascular endothelial growth factor-C (VEGF-2) has shown physiologic and anatomic evidence of angiogenesis both in animal models and clinical trials for myocardial ischemia. A large phase IIb clinical trial (the Genasis trial) is ongoing to evaluate the safety and efficacy of pVGI.1 (VEGF2) for treatment of severe cardiovascular disease. We report here a nonclinical study to examine biodistribution of the pVGI.1 (VEGF2) plasmid DNA following direct injection via the StilettoTM Endocardial Direct Injection Catheter System into myocardium of the left ventricle of normal pigs. The study consisted of two groups with three females per group. On day 1, Group 1 received vehicle (phosphate buffered saline) and Group 2 was treated with pVGI.1 (VEGF2) at a concentration mimicking the highest dose designed in our ongoing phase IIb CAD clinical trial. Doses were administered as six distinct injections (1.0 ml at 133.3 mg/ml per injection) at 1.0ml/min flow rate into the anterior wall of the left ventricle via Stiletto™. The volume, number of injections and delivery mode were consistent with the design of the clinical trial. Tissue samples of ovary, spleen, kidney, liver, mediastinal lymph node, lung and heart were aseptically collected on the 5th day post administration. Biodistribution of pVGI.1 (VEGF2) DNA was analyzed by a very sensitive and specific q-PCR assay. This assay detects 126 base pair sequence unique to the pVGI.1 (VEGF2) without interference from the endogenous pig genomic VEGF2. The sensitivity of the assay is 5 copies per mg genomic DNA and the quantification range is from 50 to 1 X 106 copies per mg genomic DNA. The pVGI.1 (VEGF2) DNA was neither detectable in any of the tissues from the vehicle control group (Group 1) nor in ovaries, livers and lungs of treated animals of Group 2. However, pVGI.1 (VEGF2) DNA was detected in mediastinal lymph nodes, kidneys and hearts of Group 2 animals. The levels ranged from less than 50 to several hundred copies per mg genomic DNA. Detection of pVGI.1 (VEGF2) in mediastinal lymph nodes may be due to their proximity to the heart. There is a possibility that degraded DNA fragments in circulation were gradually eliminated through kidney. A qRT-PCR assay is currently under development and will be used to examine whether there is any unintended VEGF2 expression in non-target organs. In conclusion, we have not observed pVGI.1 (VEGF2) dissemination to gonads nor to most of non-target vital organs and hence there is no risk of germ line transmission at day 5 post intramyocardial administration.
S358
926. Recombinant AAV Vectors Pseudotyped with Serotype-1 Capsid Mediates Early Onset of Gene Expression in Mouse Hearts Hua Su,1 Yu Huang,1 Alicia Barcena,2 Janice Arakawa-Hoyt,1 Jianqin Ye,3 William Grossman,1,3 Yuet W. Kan.1,2 1 Cardiovascular Research Institute, University of California, San Francisco; 2Laboratory Medicine; 3Cardiology Division, University of California, San Francisco, San Francisco, CA. Adeno-associated virus serotype 2 (AAV2) has been used most frequently as vector in experiments on cardiac gene therapy. We have used this vector to deliver vascular endothelial growth factor (VEGF) gene controlled by the hypoxia response element (HRE) and a cardiac myosin light chain 2v promoter. We achieved cardiacspecific and hypoxia-responsive gene expression that induced neovascular formation in ischemic murine hearts and improved cardiac function. However, gene expression mediated by AAV2 vector in hearts was delayed and did not reach the maximum level until about 4 weeks after intramyocardial injection. Irreversible myocardial injury could have occurred during this period of delay. Because hypoxia inducible factor 1 (HIF-1) is induced immediately at the onset of hypoxia, earlier onset of the HRE regulated gene expression should be more effective in inducing therapeutic angiogenesis. Recently, studies on AAV with different serotypes have shown that they differ in their transduction efficiency with different tissues. To identify a serotype that can efficiently mediate early expression of transgene in the myocardium, we packaged into serotypes 1 to 5 capsids, AAV vector carrying CMV promoter driving the mouse erythropoietin (Epo) gene. The packaged vectors were injected into murine hearts intramyocardially. Gene expression was studied by real-time RT-PCR. The kinetics of Epo expression was studied by measuring the hematocrit after AAVEpo gene transduction at different time points. We found that AAV1, 4 and 5 mediated earlier and higher gene expression than AAV2. Gene expression mediated by AAV1 increased faster than AAV4 and 5, and became the highest after day 4. To compare the early gene expression mediated by AAV serotype 1 and 2, we packaged into these capsids AAV-LacZ vectors that carried a CMV promoter driving LacZ gene expression and injected them to normal and ischemic hearts. Gene expression was detected by LacZ staining. The LacZ expression was detected in the normal and ischemic myocardium one day after AAV1-LacZ injection, while no LacZ expression could be detected in the hearts injected with AAV2-LacZ at this time. At day 14, 100% of the cardiomyocytes around needle injection sites were infected by AAV1-LacZ vector, whereas only sporadic cardiomyocytes were infected by AAV2LacZ. These results indicate that AAV1 can mediate gene expression within one day. HRE regulated VEGF gene packaged in this capsid will induce very early gene expression and therefore enhance the therapeutic effect.
927. Human Mesenchymal and Hematopoietic Stem Cell Contribution to Cardiac Tissue Regeneration after Injury Ivana Rosova,1 Jan A. Nolta.1 Internal Medicine/Oncology/Stem Cell Biology, Washington University in St. Louis, St. Louis, MO.
1
Cardiomyocyte regeneration by stem cells from non-cardiac tissues could be a future treatment for cardiovascular diseases. However, there is little direct evidence that adult stem cells can regenerate a damaged heart. In vitro assays of stem cells from the bone marrow compartment using 5-azacytidine suggests that these stem cells can adopt the cardiomyocyte morphology and express cardiac-specific markers. One of the most commonly used in vivo models for myocardial infarction is coronary ligation. In this model, the left anterior descending (LAD) coronary artery is tied off to create an Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
CARDIOVASCULAR: MYOCARDINAL GENE THERAPY area of ischemia in the left ventricle. Using this model, it remains a controversy as to whether stem cells from the bone marrow, either hematopoietic or mesenchymal, can contribute and/or enhance cardiomyocyte regeneration following damage. Our lab has previously reported that co-transplantation of hematopoietic stem cells (HSC) with mesenchymal stem cells (MSC) greatly enhances the complete hematopoietic lineage reconstitution in myeloablated immunodeficient mice. To address the role of HSC and/or MSC in repair for cardiovascular diseases, we have developed a heart injury model using doxorubicin, a chemotherapeutic drug that has been shown to cause cardiac toxicity. Hematopoietic and mesenchymal cells are injected next to the ischemic area immediately after the LAD surgery, or intraperitoneally/intravenously two days after the doxorubicin injection. LAD-injured mice are then assessed functionally by echocardiography, to obtain ejection fraction, end systolic volume, end diastolic volume and wall motion score indexes. Our preliminary studies showed show low but persistent engraftment of human cells in the cardiac tissue in 100% of surviving transplants. An additional aim of our studies is to ask whether cotransplantation of HSC and MSC could give a functional phenotype suggestive of cardiovascular repair. To distinguish HSC from MSC following their co-transplantation, we transduced HSC with a retroviral construct expressing YFP and MSC either with a monocistronic retroviral construct expressing GFP alone or with a bicistronic retroviral construct expressing a chemotatic/proliferation factor (hepatocyte growth factor) and GFP. The use of the bicistronic construct is to determine whether secretion of HGF in vivo might enhance the survival of either HSC, MSC, or both, in the damaged heart. Moderate but detectable improvements in cardiac function can be seen in these mice. Whether the exogenous human HSC and/ or MSC had transdifferentiated into cardiomyocyte-like cells or whether the injected cells indirectly recruit endogenous cardiomyocyte stem cells to proliferate and repair the cardiac damage is currently being investigated. Collectively, the use of retroviral gene transfer of different fluorescent markers to track stem cells in vivo in this cardiac-damaged xenotransplantation model will provide a more definitive answer as to which stem cells of the bone marrow compartment enhance cardiac repair. The use of retroviral gene transfer of specific soluble factors, such as HGF, could provide additional survival and/or differentiation support of stem cells in vivo.
928. Nonclinical Safety Study of Direct Myocardial Injection of pVGI.1 (VEGF2) Using the Stiletto™ Endocardial Direct Injection Catheter System in a Swine Model Qing Wang,1 Robert Rapoza,2 Wendy Naimark,2 Hsing-Yi Jan,1 Yawen L. Chiang.1 1 Preclinical, Corautus Genetics, Inc., San Jose, CA; 2Boston Scientics Inc., Natick, MA. Intramyocardial delivery of vascular endothelial growth factor-C (VEGF-2) has shown physiological and anatomic evidence of angiogenesis in animal models and clinical trials for myocardial ischemia. We evaluated safety and histopathological toxicity of multiple intramyocardial injections of DNA plasmid encoding vascular endothelial growth factor C, pVGI.1 (VEGF2) via the Boston Scientific StilettoTM Endocardial Direct Injection System (Stiletto™) in normal Yorkshire swine. The study consisted of two groups (3 males and 3 females per group). On day 1, Group 1 animals received vehicle (phosphate buffered saline) and Group 2 were treated with pVGI.1 (VEGF2) plasmid at a concentration mimicking the highest dose designed in our ongoing phase IIb CAD clinical trial (the Genasis trial). Doses were administered as six distinct injections (1.0 ml at 133.3 mg/ml per injection) at 1.0ml/min flow rate into the anterior wall of the left Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
ventricle via Stiletto™ . The volume, number of injections and delivery mode were consistent with the design of the clinical trial. Blood samples were collected pre- and post-treatment for chemistry and cardiac enzyme analyses. EKG and blood pressure were continuously monitored during the interventional procedure. Clinical observations and physical examinations were performed and body weights were recorded at various time points post treatment. Animals were euthanized on day 5 and subjected to comprehensive necropsy. Heart tissues were collected for gross and histopathological evaluation. The results showed that intramyocardial delivery of six 1.0 ml injections of pVGI.1 (VEGF2) plasmid into the left anterior ventricular wall using the StilettoTM catheter was well tolerated in this five-day experiment. There were no unscheduled deaths or treatment-related adverse clinical observations. Clinical pathology assessment did not reveal any adverse effects related to either the injection procedure or the treatment. Gross evaluation of the heart, the aorta, aortic arch, and aortic valve did not identify any lesions in either group. As expected, there were mild microscopic inflammatory cell infiltrations along the needle track comparable between groups. Additional microscopic changes included fibroplasias, degeneration and regeneration of myofibers similar in morphologic appearance and intensity between the vehicle control and plasmid treated groups. In conclusion, there was no evidence of systemic or local toxicity associated with the intramyocardial administration of pVGI.1 (VEGF2) plasmid in this 5-day study of porcine model. The results presented here provided nonclinical safety data in support of our ongoing human trial.
929. CD9 Gene Therapy Inhibits Cardiac Hypertrophy and Tachycardia, and Attenuates the Remodeling after Myocardial Infarction in Mice Hiroaki Ushikoshi,1 Tomoyuki Takahashi,2 Genzou Takemura,3 Yuwen Li,3 Masayasu Esaki,1 Ngin Cin Khai,1 Takao Kawai,1 Shinya Minatoguchi,3 Takako Fujiwara,4 Hisayoshi Fujiwara,3 Ken-ichiro Kosai.2 1 Department of Gene Therapy and Regenerative Medicine, Gifu University, Gifu, Japan; 2Department of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute of Brain Diseases, Kurume University, Kurume, Japan; 3 Department of Cardiology, Regeneration & Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, Japan; 4Department of Food Science, Kyoto Women’s University, Kyoto, Japan. BACKGROUNDS: Cardiac hypertrophy is an adaptive response of the injured heart, but usually results in heart failure and sudden death. Tachycardia is accompanied with various cardiovascular diseases and is a cause of sudden death. Although many studies of cardiac gene therapy were previously reported, none of them directly targeted cardiac hypertrophy nor tachycardia, and therapeutic genes used in them mainly targeted to enhance the angiogenesis rather than to directly modulate pathologic conditions of cardiomyocytes. On the other hand, a definitive biological function of CD9, a member of transmembrane 4 superfamily, remains elusive although its multifunctional characteristics have been reported; knockout mouse studies by us and others solely revealed the crucial role of CD9 in gamete membrane fusion (Science 287, 321-324; 2000). Here we present a novel biological function of CD9, by which we develop an innovative cardiac gene therapy. METHODS AND RESULTS: (1) In vitro experiments: Primary cultured mouse ventricular cardiomyocytes, which had been beforehand infected with either an adenoviral vector expressing CD9 (Ad.CD9) or a control adenoviral vector expressing no gene (Ad.dE1.3), were stimulated by angiotensin II, heparin-binding EGF-like growth factor (HB-EGF) or HGF. The cardiomyocytes that had received any of the stimuli and Ad.dE1.3 S359
