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1187. Long-Term Regulated Gene Expression in Non-Human Primates
GENE REGULATION: REGULATED GENE EXPRESSION been improved 10-fold, large-scale production of these pseudotyped vectors are expected to be easier compared to vectors pseudotyped with the EboZ GP. James M. Wilson holds equity in Targeted Genetics, Corp.
GENE REGULATION: REGULATED GENE EXPRESSION 1187. Long-Term Regulated Gene Expression in Non-Human Primates Victor M. Rivera,1 Guang-ping Gao,2 Rebecca L. Grant,2 Lili Wang,2 Michael A. Schnell,2 Philip J. Zoltick,2 Leonard W. Rozamus,1 Tim Clackson,1 James M. Wilson.2 1 ARIAD Gene Therapeutics, Cambridge, MA, United States; 2 Division of Medical Genetics, University of Pennsylvania, Philadelphia, PA, United States. Gene therapy is an attractive general route for the delivery of secreted therapeutic proteins. However, most proteins have limited therapeutic windows, mandating the inclusion of a regulatory system that allows protein production to be pharmacologically controlled, and terminated if necessary. We have developed a regulation system in which transcription of a target gene is made dependent upon a transcription factor that is activated by rapamycin, or nonimmunosuppressive derivatives. The system is characterized by tight, dose-responsive control, and is built from human proteins to minimize potential immunogenicity in a clinical setting. We previously reported tightly regulated expression of erythropoietin (Epo) in mice upon co-injection into muscle of two AAV vectors, one constitutively expressing the regulatory transcription factors and the other encoding the target gene. As a prelude to clinical applications we have used rhesus macaques as a large animal model to analyze and optimize the delivery of rhesus Epo (rhEpo) from AAV vectors. In initial studies, six rhesus macaques were transduced with the two-vector system. Reversible rhEpo induction and elevated hematocrit were observed in all six, but inducibility did not persist beyond two cycles in three of the monkeys (∼90 days post-transduction). However, the three other animals demonstrated long-term regulated expression for the duration of the experiment, which for the longest-studied animal is currently >4.5 years. To improve system performance, we designed a secondgeneration transcription factor vector in which expression levels are significantly enhanced. All eight macaques co-transduced with this vector and a rhEpo target gene vector have demonstrated long-term regulation (>4 years for the longest-studied animals) in response to rapamycin or AP22594, an orally-bioavailable rapamycin analog with substantially reduced immunosuppressive activity. Epo levels are indistinguishable from background endogenous levels in the absence of inducer (<2 mU/ml) and are increased in a dose-dependent manner in the presence of inducer, with peak levels >1000 mU/ml observed in animals administered high vector doses. Hematocrit levels are completely reversible, further attesting to the lack of leakiness. The long duration of regulation implies that there is no cellular immune response to the components of the regulatory system. To simplify vector manufacture and delivery, and to broaden the potential target tissues beyond syncytial muscle, we have consolidated all of the components of the system into a single AAV vector. Tight, dose-responsive rhEpo regulation has been observed in a rhesus macaque administered a single AAV2 serotype vector (>10 months to date). Substantially higher rhEpo levels are induced in a macaque administered a ten-fold lower dose of an AAV1 serotype vector, demonstrating that AAV1 transduces macaque muscle much more efficiently than AAV2, as has been previously observed in mice. Together these data support the development of intramuscular administration of regulated AAV vectors for the delivery of secreted proteins. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
VMR, LWR and TC are employed by and hold equity in ARIAD Pharmaceuticals. JMW holds equity in Targeted Genetics.
1188. Long-Term Regulated Gene Expression in the Eye by Adeno-Associated Virus Gene Therapy Corinna Lebherz,1,2 Alberto Auricchio,1,2 Albert Maguire,3 Victor M. Rivera,4 Jean Bennett,3 James M. Wilson.1,2 1 Dept. of Medicine, Division of Medical Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA; 2The Wistar Institute, Philadelphia, PA; 3Dept. of Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, PA; 4ARIAD Pharmaceuticals, Inc., Cambridge, MA. A promising new option for the treatment of inherited or acquired ocular diseases is adeno-associated virus mediated gene therapy. Several studies already exist showing promising results in the treatment of retinal dystrophies and neovascularization, which otherwise might have caused blindness. In general, however, it would be useful to be able to regulate transgene expression with the aim of maximizing the therapeutic effect and minimizing toxicity. For this study a regulated bicistronic vector system integrated into an AAV vector was used, in which expression of a transgene is dependent on rapamycin-induced reconstitution of a functional transcription factor. Erythropoietin (Epo) was chosen as transgene, because it is a highly potent protein that can be repeatedly measured in eye fluids and has a readily detectable systemic effect. The vector was injected either sub-retinally or intravitreally into non-human primate eyes. Anterior chamber (AC) fluid and sera were obtained on a weekly basis to determine changes in Epo and Hct. Induction of transgene expression was achieved via intravenous administration of rapamycin at designated timepoints with declining dosages. Epo levels detected in the anterior chamber fluid did peak generally 7 to 14 days after induction and slowly declined back to baseline thereafter. Peak Epo levels did correlate to the dose rapamycin administered. There were neither increases in serum Epo nor hematocrit detected after induction.
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GENE REGULATION: REGULATED GENE EXPRESSION 1187. Long-Term Regulated Gene Expression in Non-Human Primates Victor M. Rivera,1 Guang-ping Gao,2 Rebecca L. Grant,2 Lili Wang,2 Michael A. Schnell,2 Philip J. Zoltick,2 Leonard W. Rozamus,1 Tim Clackson,1 James M. Wilson.2 1 ARIAD Gene Therapeutics, Cambridge, MA, United States; 2 Division of Medical Genetics, University of Pennsylvania, Philadelphia, PA, United States. Gene therapy is an attractive general route for the delivery of secreted therapeutic proteins. However, most proteins have limited therapeutic windows, mandating the inclusion of a regulatory system that allows protein production to be pharmacologically controlled, and terminated if necessary. We have developed a regulation system in which transcription of a target gene is made dependent upon a transcription factor that is activated by rapamycin, or nonimmunosuppressive derivatives. The system is characterized by tight, dose-responsive control, and is built from human proteins to minimize potential immunogenicity in a clinical setting. We previously reported tightly regulated expression of erythropoietin (Epo) in mice upon co-injection into muscle of two AAV vectors, one constitutively expressing the regulatory transcription factors and the other encoding the target gene. As a prelude to clinical applications we have used rhesus macaques as a large animal model to analyze and optimize the delivery of rhesus Epo (rhEpo) from AAV vectors. In initial studies, six rhesus macaques were transduced with the two-vector system. Reversible rhEpo induction and elevated hematocrit were observed in all six, but inducibility did not persist beyond two cycles in three of the monkeys (∼90 days post-transduction). However, the three other animals demonstrated long-term regulated expression for the duration of the experiment, which for the longest-studied animal is currently >4.5 years. To improve system performance, we designed a secondgeneration transcription factor vector in which expression levels are significantly enhanced. All eight macaques co-transduced with this vector and a rhEpo target gene vector have demonstrated long-term regulation (>4 years for the longest-studied animals) in response to rapamycin or AP22594, an orally-bioavailable rapamycin analog with substantially reduced immunosuppressive activity. Epo levels are indistinguishable from background endogenous levels in the absence of inducer (<2 mU/ml) and are increased in a dose-dependent manner in the presence of inducer, with peak levels >1000 mU/ml observed in animals administered high vector doses. Hematocrit levels are completely reversible, further attesting to the lack of leakiness. The long duration of regulation implies that there is no cellular immune response to the components of the regulatory system. To simplify vector manufacture and delivery, and to broaden the potential target tissues beyond syncytial muscle, we have consolidated all of the components of the system into a single AAV vector. Tight, dose-responsive rhEpo regulation has been observed in a rhesus macaque administered a single AAV2 serotype vector (>10 months to date). Substantially higher rhEpo levels are induced in a macaque administered a ten-fold lower dose of an AAV1 serotype vector, demonstrating that AAV1 transduces macaque muscle much more efficiently than AAV2, as has been previously observed in mice. Together these data support the development of intramuscular administration of regulated AAV vectors for the delivery of secreted proteins. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
VMR, LWR and TC are employed by and hold equity in ARIAD Pharmaceuticals. JMW holds equity in Targeted Genetics.
1188. Long-Term Regulated Gene Expression in the Eye by Adeno-Associated Virus Gene Therapy Corinna Lebherz,1,2 Alberto Auricchio,1,2 Albert Maguire,3 Victor M. Rivera,4 Jean Bennett,3 James M. Wilson.1,2 1 Dept. of Medicine, Division of Medical Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA; 2The Wistar Institute, Philadelphia, PA; 3Dept. of Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, PA; 4ARIAD Pharmaceuticals, Inc., Cambridge, MA. A promising new option for the treatment of inherited or acquired ocular diseases is adeno-associated virus mediated gene therapy. Several studies already exist showing promising results in the treatment of retinal dystrophies and neovascularization, which otherwise might have caused blindness. In general, however, it would be useful to be able to regulate transgene expression with the aim of maximizing the therapeutic effect and minimizing toxicity. For this study a regulated bicistronic vector system integrated into an AAV vector was used, in which expression of a transgene is dependent on rapamycin-induced reconstitution of a functional transcription factor. Erythropoietin (Epo) was chosen as transgene, because it is a highly potent protein that can be repeatedly measured in eye fluids and has a readily detectable systemic effect. The vector was injected either sub-retinally or intravitreally into non-human primate eyes. Anterior chamber (AC) fluid and sera were obtained on a weekly basis to determine changes in Epo and Hct. Induction of transgene expression was achieved via intravenous administration of rapamycin at designated timepoints with declining dosages. Epo levels detected in the anterior chamber fluid did peak generally 7 to 14 days after induction and slowly declined back to baseline thereafter. Peak Epo levels did correlate to the dose rapamycin administered. There were neither increases in serum Epo nor hematocrit detected after induction.
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