from Herpes simplex virus suitable for human cancer gene therapy. J. HLAVATÝ, К. ... In some cases, existence of a powerful bystander effect may partially ...
NEOPLASMA, 46, б, 1999
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Cells producing recombinant retrovirus with thymidine kinase gene from Herpes simplex virus suitable for human cancer gene therapy J. HLAVATÝ, К. HLUBINOVÁ, J. BiES, Č. ALTANER
Cancer Research Institute, Slovak Academy of Sciences, S33 91 Bratislava, Slovak Republic Received July 27,1999 Therapeutic cells producing amphotropic retrovirus, which are able to transduce in vivo thymidine kinase gene of Herpes simplex virus were prepared. Single-cell clone cells with high virus productivity (PA-3 17.TH5cll3) were obtained by cell cloning. The cells were found free of replication competent retrovirus, they were non-tumorigenic in xenogeneic host and highly sensitive to ganciclovir treatment in vitro and in vivo. The therapeutic efficacy of PA-3 17JH5c] 13 cells was tested in rat brain tumor model. Increase in survival in the group of treated versus untreated rats was observed. Therefore, these cells are suitable for application in human clinical trial. Key words: Retroviral vector producing cells, gene therapy, HSV/Á'.
Replication-defective retroviral vectors are widely used in gene therapy clinical trials both ex vivo and in vivo [3, 14, 15, 18, 19, 23]. Using the property of retroviruses to infect and to integrate only proliferating cells, retroviral vectors are espe cially suitable for transfer of foreign genes into such struc tures, in which the tumor cells undergoing rapid division are surrounded by nonproliferating tissue. Among the most widely used suicide gene in cancer gene therapy both in clinical and preclinical studies is the thymi dine kinase gene of Herpes simplex virus type 1 (HSWc). Transfer and expression of HSV//c gene into tumor cells con fers sensitivity of these cells to the antiviral drugs such as ganciclovir (GCV). HSV tk efficiently and specifically phosphorylates GCV into monophosphorylated form, which is further metabolized by cellular kinases to ganciclovir-triph osphate, an inhibitor of DNA polymerase and chain terminator. There are several shortcomings connected with the use of retrovirus-producing cells as gene therapy tools. Beside oth ers, the necessity to produce large amounts of therapeutic cells producing retrovirus might have caused derivation of low vi rus-producing cells in the cell population used for clinical
*Tlic study was supported by the Grant No. 2/4028/98 from the VEGA Grant Agency.
application [4]. Therefore, the cells used in human gene ther apy trials should be either checked for their stability of virus production, or precautions have to be done to avoid this ob stacle. However, a major limitation of gene therapy for cancer at present is the limited ability to transduce large amount of ther apeutic cells into the tumor mass. Using retroviral vectors usually only about five percent of cells are transduced in vivo. In some cases, existence of a powerful bystander effect may partially overcome the limitation of inefficient gene trans duction. The bystander effect in suicide gene therapy is de scribed as killing of non-transduced cells growing in a mixture with transduced cells after prodrug administration and was observed both in vitro and in vivo. In the case of HSVtk gene and ganciclovir as a prodrug, tight cell-to-cell contact is re quired for efficient bystander effect. The mechanism of by stander effect is not fully understood. However, it was report ed that connexin expression and cell communication via gap junctions play a cardinal role in the transfer of phosphorylated GCV between cells [2, 29]. Other mechanisms, such as transfer of apoptotic bodies or involvement of an immune component in vivo may also be involved [9—12]. In this study the preparation of HSV tk gene containing retrovirus producer cells PA-3 17,1 H5cl 13 that might be po tentially used in human gene therapy clinical trials is described.
HLAVATÝ, HLUBINOVÁ, BIES. ALTANER
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The therapeutic recombinant retrovirus is free of replicationcompetent retroviruses and is produced from single cell clone with high viral titer. The effect of GCV treatment on survival of rats with established brain tumors inoculated with thera peutic virus producing cells was studied in vivo. Material and methods Cell lines. Cells were maintained at 37'C in a humidified air atmosphere containing 5% C 0 2 . Murine fibroblast retro viral packaging cell line PA-317 [22], mouse fibroblast NIH 3T3/4070A cells producing the 4070A strain of amphotropic murine leukemia virus (MLV) [21] and Muss duni tail fibro blast cells [17] were cultivated in Dulbecco's modified Ea gle's medium (DMEM) supplemented with 10% heat-inacti vated fetal calf serum (PCS), glutamine and antibiotic mix (Sigma, St. Louis, MO, USA). Rat glioma cell line C 6 and human cervical carcinoma cell line HeLa were cultivated in DMEM containing 5% PCS, glutamine and antibiotic mix. PG-4 S+L- cat cells [1] were cultured in McCoy's 5A medi um supplemented with 15% PCS and antibiotic mix. Generation of virus producing cells. Construction of the retroviral vectorpJH5 was described earlier [13]. To generate virus producing cells, packaging cell line PA-317 was transfected with 15 (Xg of pJH5 DNA using DOTAP transfection reagent (Boehringer-Mannheim, FRG) according to manu facturer's recommendation. Two days later, cells were seeded in the medium containing 0.4 mg/ml of G418 and selected until the colonies were grown up. Single colonies were picked up using cloning rings, cells were expanded, and reverse tran scriptase activity was determined in medium from these col onies. The colony that gave the highest activity of reverse transcriptase was subjected to single cell cloning by limited dilution. Medium from single-cell clones was again checked by reverse transcriptase assay. Clones with the highest activ ity were further characterized. Reverse transcription assay. 50 u.1 of virus containing cellfree medium (either fresh or frozen at -70'C) was mixed with 10 ul of reaction mixture [(0.02% Nonidet P40, 1 mol KCl, 0.1 mol MnCl,, 10 mmol dATP, 10 mmol dGTP, 10 mmol dCTP, 20 mmol Tris-HCl pH 8, 0.4 mmol EDTA pH 7.5, 10 mmol DTT, 74 k B q 3 H d T T P and 0.2 ug poly(rA) oligo(dT)]. The incubation was performed at 37'C. 15 ul aliquots in three time points (0,30, and 60 minutes of incubation) were dropped onto Whatman 541 paper discs, dried, and washed for 15 min utes in 10% trichloracetic acid (TCA) with 1% N a 4 P 2 0 7 then twice for 10 minutes in 5% TCA followed by final washing for 10 minutes in ethanol. All washing steps were performed on ice. After the ethanol washing, discs were dried and radio activity was measured by scintillation counter. Viral titer and infection. To estimate the titer of virus pro ducing cells, 2 x 10 5 of HeLa cells were plated in 60 mm
Petri dish one day before infection. Virus-producing cells were grown in medium until they reached confluence and the cul ture fluid was replaced. Twenty-four hours later, virus-con taining medium was collected and filtered through a 0.45\im filter. Infection was done by replacing the medium of the recipient cells by 2 ml of virus-containing medium (either diluted or undiluted) in the presence of polybrene (8 ug/ml). After 24 hours, medium was changed for fresh medium con taining 1.0 mg/mľof G418 for HeLa cells. Selection was al lowed to proceed for 10—12 days. Medium was changed as necessary during this selection process. Macroscopic colo nies were stained with crystal violet and counted. +
5
To prepare C 6 T K cells, 5 x 10 C 6 cells were infected with retrovirus containing medium in the presence of polybrene (8 (J.g/ml). Two days later, cells were selected in 0.4 mg/ml of G418 for two weeks. Colonies of infected cells were pooled and used in further experiments. Replication-competent retrovirus (RCR) detection. To de tect the presence of RCR in virus producing cells, method described by PRINTZ et al. [26] was used. Briefly, to amplify the prospective RCR, therapeutic cells were co-cultivated with Muss duni cells in one to one ratio in medium containing po lybrene (4 u.g/ml). The cells were passaged two times in the ratio one to ten every three days. Then, the medium was col lected, passaged through 0.45-pn filter and used to infect PG-4 S+L- cells in the presence of polybrene (4 |ig/ml). Four days later, infected PG-4 S+L- cells were evaluated for focusforming formation. As a positive control, various dilutions of medium from NIH 3T3/4070A cells, producing amphotropic MLV were used in the same manner except for the amplification step on Muss duni cells. GCV sensitivity assay. To measure the sensitivity of cells to ganciclovir, cells were plated at density 2 x 10 3 cells per well in 96-well flat-bottomed plates. Twenty-four hours later, cells were cultured with 200 \x\ of fresh medium containing differ ent concentrations of ganciclovir (Cymevene, Syntex Pharm AG, Basel, Switzerland). Three days later, medium was changed and after 5 days of incubation with GCV, medium was removed and cytotoxicity was determined using CellTiter 96°-°AQucous_ Non-Radioactive Cell Proliferation Assay (Promega Co, Madi son, WI, USA) according to manufacturer's recommendation. In vivo experiments. To study the growth characteristics of producer cells in vivo, the following experiments were done: BALB/c-nu nude mice were subcutaneously inoculated with 10 6 of PA-317JH5cl 13 cells. When the cell mass reached ap proximately 10 mm in diameter, mice were randomly divided into two groups. One group received GCV treatment (50 mg/ kg) twice a day for five days, starting thirteen days after in oculation. Second group was used as untreated control. In a xenogeneic model Sprague-Dawley rats were used. Either five or ten days old rats were subcutaneously injected with 5 x 10 6 or 10 6 of PA-317JH5cll3 cells. The animals were observed during one month.
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To test the therapeutic effect of prepared virus producing cells, adult Sprague-Dawley rats of 180—220 g weight were used. Rats were anesthetized (atropine 0.1 mg/kg, rometar 10 mg/kg, narkamon 90 mg/kg) and stereotactically injected with 5 x 104 of C 6 cells or 5 x 105 of C 6 TK + cells re-suspended in 5 pi of TD (о'. 1 mol Tris buffer pH 7.4 without Ca++ and Mg++) into the caudate nucleus 3 mm lateral to the bregma and 5.5 mm deep. Injections were carried out over two minutes using a 10 (il Hamilton syringe. The needle was kept for another 2 minutes in the site of inoculation. Four days after intracere bral inoculation of C 6 cells, 107 of PA-317JH5cll3 virus pro ducing cells resuspended in 50 p.] of TD were inoculated us ing the same coordinates. The injection of therapeutic cells was done over 5 minutes period and the needle was kept for another 5 minutes. Ganciclovir treatment at a dosage 25 mg/ kg of body weight twice a day for two weeks was started 4 days after the virus-producing cell inoculation. The animals were inspected daily and moribund rats were killed and the brain was analyzed histologically.
Cells designated as PA-317JH5cll3 produced the highest amount of infectious virus (2.55 x 106 cfu/ml of infectious medium) and were used in experiments throughout. To further characterize these cells, their sensitivity to ganciclovir was estimated. Cells were found to be highly sensitive to GCV with LD 50 0.1 (ig/ml (Fig. 1).
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In our previous report construction of bicistronic, Molo ney MLV-derived retroviral vector containing Herpes simplex virus thymidine kinase gene under control of viral LTR se quence was described. In addition, single-cell virus produc ing clone that generated recombinant retrovirus at the titer 6.5 x 104 cfu/ml of virus containing medium was isolated [13]. It was shown that various single cell clones isolated from the mass culture differed in their titers as well as in their sen sitivity to GC V. In order to prepare virus producing cells gen erating recombinant retroviruses in amounts suitable for po tential use in human gene therapy, mouse fibroblast packag ing cell line PA-317 was transfected with pJH5 plasmid and different single cell clones were isolated. Based on the results of RT-activity, several clones were further tested for produc tion of infectious virus by titration on HeLa cells (Table 1).
Table I. Properties of individual single-cell clone of retrovirusproducing cells Clone
'RT(cpm)
3 9 13 19
R2R72 104148 111600 71739
5 -Titer x I0
LD s „ofGCV(üg/ml)
1.2 7 25.5 5
N.T. 1.5 0.1 N.T.
1 - cpm of [-'H]thymidine triphosphate incorporated into 0.0125 ml of tissue culture fluid incubated at 37°C for 60 min with poly(rA) oligo(dT) as a primer-template. 2 - 2 ml of overnight cell-free medium containing 8.0 ug/ml of polybrcne were used to infected HeLa cells. 24 hours later the medium was changed for medium containing G418 at a dose 1.0 mg/ml and the colonics were counted 6 to 10 days later. N.T. - not tested.
0.4
0.8
1.2 1.6 2 2.4 ganciclovir (/ug/ml)
2.8
3.2
Fig. 1. Ganciclovir sensitivity of the PA-317JH5cll3 cells.
The stability of virus production during the long-term cultivation in vitro was checked by RT assay. Generally it was found that the virus production has a trend to be lower in higher cell passages. To avoid this development, the single-cell clone derived cells in early passages were frozen at the beginning to allow starting the cell cultivation from these original cells later. The use of retroviral vectors for transfer of genetic information could be coupled with the possibility of generation of a replication-competent retrovirus (RCR). Recombination between short homologous regions within the retroviral vector and helper sequences in the packaging cells theoretically may lead to arising of RCRs. The S+L- assay is routinely used for detection of RCR in clinical batches of virus producing cells. To exclude presence of replication-competent retroviruses in PA-317JH5cll3 producer cells we used the smallscale co-cultivation assay [26]. The sensitivity of PG-4 S+Lcells for detection of amphotropic 4070A virus by focus formation was at the 10"7/ml virus dilution. No focuses were observed in PG-4 S+L- cells infected with virus-containing medium from PA-317JH5cll3 cells, which was amplified through Muss dani cells. In addition negative data were obtain in the experiment, where the RT assay was used for testing of retrovirus presence in medium from PA-317.IH5cll3 cells after amplification in the Muss cluni cells. Therefore, the therapeutic recombinant retroviruses generated from PA317JH5cll3 cells were RCR free.
HLAVATÝ, HLUBINOVÁ, BIES, ALTÁNEK
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To test the growing potential of PA-317JH5cll3 cells in vivo, two types of experiments were performed. In the first one, 106 of retroviral producer cells were injected subcutaneously into nude mice. Thirteen days after inoculation when cell mass reached an average of 10 mm in diameter, GCV (50 mg/kg of body weight) was administered twice a day for five consecutive days. Reduction in cell mass was observed dur ing the treatment period. On day fourteen, after GCV admin istration, no cell mass was detectable by palpation in treated mice. No re-growth of cells was observed until mice were sacrificed (forty-one days after virus producing cells inocu lation), demonstrating the safety of using live retroviral pro ducer cells in vivo. In the group of untreated mice, cell mass grew rapidly and all animals must have been sacrificed with in twenty-seven days after inoculation of producer cells. In the second type of experiments, either 106 of producer cells were injected subcutaneously into eleven days old SpragueDawley rats or 5 x 106 of producer cells were injected subcu taneously into five days old Sprague-Dawley rats and their growth chracteristics were tested. Neither in the first, nor in the second group of rats the cells formed palpable mass over one month. Therefore, the cells were not tumorigenic in xe nogeneic host. Experiments were performed to test the ability of prepared virus-producing cells to kill tumors transplanted into brain of rats. Totally, thirty adult rats were inoculated intracerebrally with C 6 glioma cells. Four days later 107 of PA-317JH5cll3 virus producing cells were inoculated by the same way. The GCV application at a dose 25 mg/kg twice a day for two weeks started four days later. The average survival of totally eighteen GCV treated animals was twenty-eight days com pared to twenty days for twelve animals without GCV appli cation (Table 2). In the next experiment, C ň TK + cells were
Table 2. Survival of rats after inoculation of Ck glioma cells into the brain followed by HSV/A7GCV treatment Inoculated cells QTK (5x 10') C,,TK' (5x 105) C,,(5x I01) and РЛ-3 17.1Ы5с113 (H)7) C,,(5x l0 4 )andPA-3l7JH5cll3(l0 7 ) С,, (5x ID4) and PA-317JH5cll3 (107) C,, (5 x Ю4) and PA-3 17JH5cl 13(10 ; )
GCV +
+ +
Survival (days) 23 > 210 17 exp. 1 24 cxp.2 27 exp. I 30 exp.2
used to induce gliomas in rats. The same schedule as before, except for the injection of virus-producing cells, was used. Seven out of nine rats treated with GCV were sacrificed more than six month after inoculation of C6TK7 cells without signs of any disease, two rats died at day 65 and 86 after inocula tion of C 6 TK + cells, respectively. Control animals (n= 5) with no GCV treatment survived approximately 23 days.
Discussion The recombinant retrovirus particles are produced by PA317 cells, which were derived from mouse NIH 3T3 cell line by MILLER and BUTTIMORH [22]. The viruses have an ampho-
tropic host range and therefore, can infect a variety of cell species, including human. The PA-317 cells are routinely used to package recombinant retroviruses for human gene therapy clinical trials [5, 18, 19]. High levels of connexin 43 and high activity in gap junctional intracellular communication were found in PA-317 packaging cell line [20] and might represent an advantage in the safety of using live retroviral producer cells in vivo with HSV'/c/GCV suicide strategy. In the gene therapy protocols, packaging cells are usually injected into the tumor, or in the case of brain tumors virus producing cells are inoculated into the cavity after extirpa tion of the tumor mass [14, 16, 27]. The inoculated producer cells may continue to release therapeutic retrovirus for an extended period, thus infecting more cells as compared to inoculation of virus alone. To exclude the possible risk of uncontrolled growth of PA-317JH5cll3 cells in vivo, the growth characteristics in syngenic and xenogeneic hosts were evaluated. In experiments performed on nude mice it was shown that although virus-producing cells are able to grow on syngenic hosts, they are quickly and completely killed when GCV is administered. Therefore, the therapeutic cells are safe for clinical application. Producer cells were injected subcutaneously into young Sprague-Dawley rats and their growth chracteristics were test ed. No growing cell mass was detected even when 5 x 106 of cells were inoculated. In this model of xenogeneic transfer from mice to rats cells should be killed by means of natural antibodies and complements. Both kinds of immune response are not actually affected by the conditions of recipient's im mune system. This observation is in agreement with those published by others as follows. When psi 2-BAG packaging cells producing recombinant retrovirus expressing E. coli lacZ gene were grafted into the rat brain, virus producing cells were detected one day after inoculation, but not five days af ter inoculation. This suggests that grafted cells were com pletely rejected [28]. However, using in situ hybridization, survival of vector producing cells at seven days after inocula tion in patients treated with HSVfTc/GCV gene therapy ap proach for recurrent malignant brain tumors was demonstrat ed [27]. Much discussion has taken place on the safety of retroviral vectors used in gene therapy, especially on the generation of replication-competent retroviruses. Recombination in the vi rus producer cell line between the genetic elements encoding the retroviral structural proteins and the vector sequence might have lead to generation of replication-competent retroviruses [24]. The administration of large amounts of virus producing cells in clinical trials represents the greatest potential expo-
CliLLS PRODUCING RECOMBINANT RETROVIRUS
333
sure of patients to R C R . T h e r e f o r e , testing of virus p r o d u c i n g
E., BENAZZI, E., BERNARDI, M., PORTA, F., FERRARI, G., Мл-
cells prior to their use in patient t r e a t m e n t is inevitable. To
viLio, F., ROSSINI, S., BLAESE, R.M., CANDOTTI, F.: Transfer of
exclude
the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogenic bone marrow transplantation. Hum. Gene Then, 6, 1995,813—819.
t h e p r e s e n c e o f R C R i n p r e p a r e d c e l l s , РА-
3 1 7 J H 5 c l l 3 p r o d u c e r cells w e r e tested in P G - 4 S + L - focusf o r m i n g assay a c c o r d i n g to U.S. Food and D r u g A d m i n i s t r a tion ( F D A ) r e c o m m e n d a t i o n s [30]. N o R C R s w e r e p r o d u c e d out o f t h e s e cells. Several studies have s h o w n that m u r i n e
[4]
R C R s cause no p a t h o l o g y in n o n h u m a n p r i m a t e s [6, 7]. How
Association of U-87 cell death with ganciclovir-mediatcd apoptosis of nearby cells and lack of effect in athymic mice. Hum. Gene Then, 6, 1995, 763—772.
ever, in an e x p e r i m e n t u s i n g s e v e r e l y i m m u n o s u p p r e s s e d R h e s u s m o n k e y , three o u t o f n i n e a n i m a l s d e v e l o p e d lym p h o m a a n d died after a d m i n i s t r a t i o n o f ex vivo t r a n s d u c e d
[5]
b o n e m a r r o w p r o g e n i t o r cells e x p o s e d to high doses o f R C R Evaluation a n d R e s e a r c h ( C B E R ) o f the U.S. F D A r e c o m m e n d s to m o n i t o r patients for R C R occurrence. In recent study [6]
R.M., ANDERSON, W . F : Amphotropic murine leukemia ret rovirus is not an acute pathogen for primates. Hum. Gene Then, /, 1990, 15—30.
s u m m a r i z e d and in situ g e n e r a t i o n o f R C R w a s not detected in any of evaluated patient [19]. [7]
g e n e therapy. It m i g h t b e d u e to low virus multiplicity caused
CORNETTA, K., MORGAN, R.A., GILLI, A., STRURM, S., BAL-
TRUCK.I, L., O'REILLY, R., ANDERSON, W . F : No retroviremia or pathology in long-term follow-up of monkeys exposed to a murine amhotropic retrovirus. Hum. Gene Then, 2, 1991, 215—219.
by low p r o d u c t i o n o f virus from t h e r a p e u t i c cells, w h i c h have to u n d e r g o m a n y m u l t i p l i c a t i o n in o r d e r t o o b t a i n n e e d e d a m o u n t o f cells in clinical application. T h i s can b e o v e r c o m e by p r e p a r i n g sufficient n u m b e r o f cells, w h i c h started from
CORN ETTA, K., MOEN, R.C., CULVER, K., MORGAN, R.A., MCLACHLIN, J.R., STURM, S., SELEGUE, J„ LONDON, W., BLAESE,
ceiving intracerebral inoculation of virus p r o d u c i n g cells were
Low t r a n s d u c t i o n efficiency is the limiting step in in vivo
COOPER, D., PENNY, R., SYMONDDS, G., CARR, A., GERLACH,
W., SUN, L.Q., ELY, J.: A marker study of therapeutically transduced CD4+ peripheral blood lymphocytes in HTV dis cordant identical twins. Hum. Gene Then, 10, 1999, 1401 — 1421.
and retroviral v e c t o r [8]. Therefore, t h e C e n t e r o f Biologic
results o f m o r e t h a n 1200 s p e c i m e n s from 128 patients re
COLOMBO, B.M., BENEDETTI, S., OTTOLENGHI, S., MORA, M., POLLO, В., POLI, G., FINOCCHIARO, G.: The "bystander effect":
[8]
DONAHUE, R.E., KESSLER, S.W., BODINE, D., MCDONAGH, K.,
the frozen stock of highly virus p r o d u c i n g cells o f the origi
DUNBAR, C , GOODMAN, S., AGRICOLA, В., BYRNE, E., RAFFLED,
nal single-cell c l o n e . It is also e x p e c t e d that single injection
М., MOEN, R., BACHER, J., ZSEBO, K.M., NIENHUIS, A.W.:
Helper virus induced T cell lymphoma in non-human pri mates after retroviral mediated gene transfer. J. Exp. Med., 176, 1992, 1125—1135.
of virus p r o d u c i n g cells w o u l d lead only to t r a n s d u c t i o n of small p o r t i o n o f cells. It could b e anticipated that repeated injections of virus p r o d u c i n g cells m i g h t result in infection o f a larger a m o u n t o f t u m o r cells. E x p e r i m e n t s were d o n e with
[9]
C.H.,
multiple injections o f p r o d u c e r cells e a c h followed by G C V Indeed, survival o f rats treated w i t h three doses o f p r o d u c e r [ 10]
W h e t h e r this a p p r o a c h would i m p r o v e the effectiveness in the d e s c r i b e d t h e r a p e u t i c s y s t e m r e m a i n s to b e d e t e r m i n e d .
KOEPLIN, D.S., MOOLTEN, F.L., ABRAHAM, G.N.: The
"bystander effect": Tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res., 53, 1993, 5274—5283.
t r e a t m e n t in a peritoneal c a r c i n o m a t o s i s m o d e l in rats [25]. cells w a s s u p e r i o r to single p r o d u c e r cells a d m i n i s t r a t i o n .
FREEMAN, S.M., ABBOUD, C.N., WUARTENBY, K.A., PACKMAN,
FREEMAN, S.M., RAMESH, R„ MARROGI, A.J.: Immune system
in suicide gene therapy. Lancet, 349, 1997, 2 — 3 . [11]
GAGENDEEP, S., BREW, R., GREEN, В., CHRISTMAS, S.E., KLATZMANN,D.,POSTON, G.J., KINSELLA, A.R.: Prodrug-activatedgene
Wc wish to thank Mrs. C. LATIAKOVÁ, M. JANOTÁKOVÁ, and V. FRIVALSKÁ for technical assistance.
therapy: Involvement of an immunological component in the "bystander effect". Cancer Gene Then, 3, 1996, 83—88. [12]
References
[1]
BASSIN, R.H., RUSCETTI, S., ALI, I., HAAPALA, D.K., REIN.A.:
Normal DBA/2 mouse cells synthesize a glycoprotein which interferes with MCF virus infection. Virology, 123, 1982, 139—151. [2]
Bi, W.L.. PARYSEK, L.K., WARNICK, R., STAMBROOK, P.J.: In
vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV TK retroviral gene therapy. Hum. Gene Then, 4, 1993, 725—731. [3]
BORDIGNON, C , BONINI, C , VERZĽLETTI, S., NOBILI, N., MAGC.IONI, D., TRAVERSARI, C , GIAVAZZI, R., SERVIDA, R, ZAPPONE,
HALL, S.J., SANFORD, M.A., ATKINSON, G., CHEN, S.H.: In
duction of potent antitumor natural killer cell activity by herpes simplex virus-thymidinc kinase and ganciclovir ther apy in an orthotopic mouse model of prostate cancer. Canccr Res., j & 1998. 3221—3225. [ 13]
HLAVATÝ, I , FILUBINOVA, К., ALTANEROVA, V, LIŠKA, .1., ALTAN-
ER, С.: Treatment of rat gliomas with recombinant retrovirus harboring Herpes simplex virus thymidine kinase suicide gene. Neoplasma, 44, 1997, 342—347. [14]
KLATZMANN, D , PIIILIPPON, J., VALERY, C A . , BENSIMON, G..
SALZMANN, I L . and the Study Group on Gene Therapy for Glioblastoma: Gene therapy for glioblastoma in adult pa tients: Safety and efficacy evaluation of an in situ injection of recombinant retrovirus-producing cells carrying the thy midine kinase gene of the Flerpes simplex type I virus, to
HLAVATÝ. HLUBINOVÁ, BIES, ALTANER
3.14
be followed with the administration of ganciclovir. Hum. GeneTher., 7, 1996, 109—126. [15]
KLATZMANN,
D.,
CHERIN,
P., BENSIMON,
G.,
MILLER, A.D., BUTTIMORE, C : Redesign of retrovirus pack aging cell lines to avoid recombination leading to helper vi rus production. Mol. Cell. Biol., 6, 1986, 2859—2902.
[23]
OLDFIELD, E.H., RAM, Z., CHIANG, Y., BLAĽSĽ, R.M.: Intrath
BOYER,
0.,CouTELLiER, A., CHARLOTTE, R, BOCCACCIO, C , SALZMANN, I L . , HERSON, S. and the Study Group on Gene Therapy of Metastatic Melanoma: A phase TAI Dose-escalation study of Herpes simplex virus type 1 thymidine kinase "suicide" gene therapy for metastatic melanoma. Hum. Gene Then, 9, 1998, 2585—2594. [16]
[22]
ecal gene therapy for the treatment of leptomeningeal carci nomatosis. Hum. Gene Ther., 6, 1995, 55—85. [24]
terization of replication-competent retrovirus resulting from recombination of packaging and vector sequences. Hum. GeneTher., 5, 1994,567—575.
KLATZMANN, D., VALERY, C A . , BENSIMON, G., MARRO, В., BOYER, О., MOKIITARI, К., DIQUET, В., SALZMANN, I L . , PIIIL-
IPPON, J. and the Study Group on Gene Therapy for Glioblas toma: A phase Т/1Г study of Herpes simplex virus type 1 thy midine kinase "suicide" gene therapy for recurrent glioblas toma. Hum. GeneTher., 9, 1998, 2595—2604. [17]
[ 18]
LANDER, M.R., CHATTOPADHYAY, S.K.: A M. dunni cell line that lacks sequences closely related to endogenous murine leukemia viruses and can be infected by ecotropic, amphotropic, xenotropic, and mink cell focus-forming viruses. J. Virol., J2, 1984,695—698. LINK, C . I , MOORMAN, D., SEREGINA, Т., LEVY, IP., SCHABOLD,
[25]
DEETROZ, В., GIELEN, I , FILLET, G., MERVILLE, M.-R, BOURS,
[26]
[27]
[20]
L.M., К ATZ, D D . , BLAESE, R.M., OLDFIELD, E.H.: Therapy of
malignant brain tumors by intratnmoral implantation of ret roviral vector-producing cells. Nat. Med., 3, 1997, 1354— 1361. [28]
[21]
SHORT, M.R, CHOI, B.C., LEE, J.K., MALICK, A., BREAKEFIELD,
X.O., MARTUZA, R.L.: Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line. I Neurosci. Res., 27, 1990, 427—439. [29]
Mc MASTERS, R., SAYLORS, R.L., JONES, K.E., HENDRIX, M.E.,
MOYER, M.R, DRAKE, R.: Lack of bystander killing in Herp es simplex virus thymidine kinasc-transduced colon cell lines due to deficient connexin 43 gap junction formation. Hum. GeneTher., P. 1998.2253—2261. MILLER, A.D., LAW, M.-R, VERMA, I.: Generation of helperfree amphotropic retroviruses that transduce a dominantacting, methotrexate-resistant dihydrofolate reductase gene. Moll. Cell Biol., 5, 1985,431—437.
RAM, Z., CULVER, K.W., OSIIIRO, E.M., VIOLA, I I , D E VROOM, H.L., OTTO, E., LONG, Z., CHIANG, Y , M c GARITTY, G.I, MUUL,
ciii, G., ENNIST, D., MARCUS, S., OTTO, E., M C GARRITY, G . I :
Biosafety monitoring of patients receiving intracerebral in jections of murine retroviral vector producer cells. Hum. GeneTher., 9, 1998, 1 165—1172.
PRINTZ, M., REYNOLDS, I , MENTO, S.I. JOLLY, D., KOWAL, K.,
SAJJADI, N.: Recombinant retroviral vector interferes with the detection of amphotropic replication competent retrovirus in standard culture assays. GeneTher., 2, 1995, 143—150.
LONG, Z., L I , L.-R, GROOMS, Т., LOCKEY, C , NADER, K.L., MYCHKOVKKY, I., MUELLER, S., BURIMSKI, I., RYAN, P., KIKU-
PRINCEN, R, LECHANTEUR, C , LOPEZ, M., ROCOUX, G.,
V: Repeated cycles of retrovirus-mediatcd HSVtk gene trans fer plus ganciclovir increase survival of rats with peritoneal carcinomatosis. GeneTher., 5, 1998, 1054—1060.
K.J.: A phase Г trial of in vivo gene therapy with the Herpes simplex thymidine kinase/ganciclovir system for the treat ment of refractory or recurrent ovarian cancer. Hum. Gene Then, 7, 1996, 1161 — 1179. [19]
OTTO, E., JONES-TROWER, A., VANIN, E. R, STAMDAUGH, K., MUELLER, S.N., ANDERSON, W.R, M C GARRITY, G . I : Charac
[30]
VRIONIS, F.D., W U , I K . , Qi, P., WALTZMAN, M., CHERINGTON,
V, SPRAY, D.D.: The bystander effect exerted by tumor cells expressing the herpes simplex virus thymidine kinase (HS Vtk) gene is dependent on connexin expression and cell com munication via gap junctions. GeneTher., 4, 1997, 5 7 7 — 585. WILSON, CA., No, T.-H., MILLER, Л.Е.: Evaluation of rec ommendations for replication-competent retrovirus testing associated with use of retroviral vectors. Hum. Gene Ther., g, 1997, 869—874.
