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401. AAV Therapy Attenuates Respiratory Dysfunction and Glycogen Accumulation in a Murine Model of Glycogen Storage Disease Type II
Musculo-Skeletal Diseases I skeletal muscles without immunosuppression resulted in insufficient transgene expression with potent immune responses. Mesenchymal stem cells (MSCs) regulate graft-versus-host disease (GVHD) by virtue of their immunosuppressive effects. Short-term exposure of MSCs with vector administration efficiently inhibited immune responses to the rAAV capsid and/or transgene products. Methods: Human bone-marrow derived MSCs and rAAV9Luciferase were intravenously injected into the normal dog at 8 weeks old. Seven days after injection, hMSCs were systemically injected again. At 8 days after 1st injection, rAAV9-Luciferase was intramuscularly injected into the tibialis anterior muscle of the same dog. To examine the immune response against rAAV, purified canine peripheral leukocytes were exposed to rAAV9 for 4 hours, and then IFN-γ expression was analyzed using qRT-PCR. Skeletal muscles of the rAAV-Luc injected animals were sampled by biopsy for expression analysis at 4 weeks after intramuscular injection. Results: Intramuscular injection of rAAV-Luc following hMSCs treatment, resulted in higher expression of Luciferase at the injected muscle, compared to the rAAV-Luc transduction alone. Expression of IFN-γ in the purified peripheral blood leukocytes after the rAAV exposure were not induced in the rAAV-Luc with MSCs, suggesting the immune suppressive effects of the MSCs. Conclusion: Our results demonstrate that rAAV-Luc injection with MSCs treatment improved expression of rAAV-derived Luciferase in dogs. This strategy would be effective approach to analyze the expression and function of transgene in vivo. These findings also support the future feasibilities of rAAV-mediated protein supplementation strategies.
401. AAV Therapy Attenuates Respiratory Dysfunction and Glycogen Accumulation in a Murine Model of Glycogen Storage Disease Type II
Darin J. Falk,1 Meghan S. Soustek,1 A. Gary Todd,1 Cathryn S. Mah,1 Denise A. Cloutier,1 Nathalie Clement,1 David D. Fuller,2 Barry J. Byrne.1 1 Pediatrics, University of Florida, Gainesville, FL; 2Physical Therapy, University of Florida, Gainesville, FL.
Cardiac dysfunction and respiratory muscle weakness are primary features in patients with early onset Pompe disease. To reduce the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa-/- mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9) or bi-monthly injections of recombinant human GAA (ERT). Cardiac function and morphology was assessed one and three months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at three months post-treatment in all groups. Gaa-/- animals receiving either AAV9 or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9 treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at three months post-injection. Neither AAV9 nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9 animals. Glycogen deposition was significantly elevated in Gaa-/- and ERT but not AAV9 when compared to control animals. These results indicate systemic delivery of either strategy improves cardiac function but improvement in respiratory parameters and glycogen reduction are limited to AAV9 therapy at three-months post treatment in a murine model of Pompe disease.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
402. Mesenchymal Stromal Cells Can Ameliorate the Progressive Phenotype of Dog With Duchenne Muscular Dystrophy
Yuko N. Kasahara,1,2 Hiromi H. Kinoh,1,2 Mutsuki Kuraoka,1 Tomoko Chiyo,1 Hironori Okada,1,2 Nana Tsumita,1 Kiwamu Imagawa,3 Katsuhiko Tachibana,3 Shin’ichi Takeda,1 Takashi Okada.1,2 1 Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan; 2 Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan; 3JCR Pharmaceuticals Co., Ltd., Ashiya, Kobe, Japan.
Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality that exhibits skeletal muscle weakness with chronic inflammation. Multipotent mesenchymal stromal cells (MSCs) could be a potential therapeutics because of their immunosuppressive properties and ability of differentiation to myogenic cells in situ. In the present study, we examined the strategies for effective cell transplantation to develop a novel approach for functional recovery of the skeletal muscles using a dog model of Duchenne muscular dystrophy. Methods: Human bone marrow-derived MSCs were intravenously injected into two littermates of canine X-linked muscular dystrophy in Japan (CXMD J ) at weekly interval for 8 weeks without immunosuppression. Clinical phenotypes on transplanted dogs were analyzed by using blood exams, physical capacity, MRI and histology compared with two non-injected littermates as controls. Results: The downregulation of inflammation in the lower legs of MSCs-treated CXMDJ was confirmed by magnetic resonance imaging analysis. Immunofluorescence analysis showed decreased area of IgG-positive necrotic myofibers and also increased number of developmental myosin heavy chain-positive regenerative fibers. Impaired tetanic force of gastrocnemius of CXMDJ recovered in hMSCs-treated CXMDJ, from 25% to 80% of wild type. Although CXMDJ showed progressive muscle atrophy in the all four limbs, exercise intolerance and abnormal locomortion, we observed improved phenotypes in the hMSCs-treated CXMDJ along with the improved pace of flip-flop and running. Conclusion: We demonstrated that the systemic injection of MSCs ameliorates the progressive phenotype in CXMDJ. The therapeutic effects might be associated with the production of paracrine or endocrine factors that regulate inflammation, and might also stimulate the proliferation of endogenous stem cells at the injured muscle tissue. This strategy of MSCs treatment would be promising for the future DMD cell therapy.
403. Engineering Micro-Dystrophin AAV Vector for Clinical Translation
Chady H. Hakim,1 Yongping Yue,1 Yi Lai,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.
1
Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy affecting boys. It is caused by the loss of a cellular structural protein called dystrophin. Dystrophin preserves the integrity of the muscle cell membrane by providing a molecular link between the cytoskeleton and the extracellular matrix. The absence of dystrophin destabilizes the muscle membrane and promotes muscle death. AAV micro-dystrophin gene therapy has transformative potentials to significantly ameliorate DMD. Unfortunately, AAV therapy is challenged by destructive cellular immunity to newly expressed dystrophin. To overcome this obstacle, we engineered a double restrictive AAV microgene vector. Specifically, we limited off-target expression in non-muscle tissue with a muscle specific promoter and removed untoward expression in antigen-presenting cells using S159
401. AAV Therapy Attenuates Respiratory Dysfunction and Glycogen Accumulation in a Murine Model of Glycogen Storage Disease Type II
Darin J. Falk,1 Meghan S. Soustek,1 A. Gary Todd,1 Cathryn S. Mah,1 Denise A. Cloutier,1 Nathalie Clement,1 David D. Fuller,2 Barry J. Byrne.1 1 Pediatrics, University of Florida, Gainesville, FL; 2Physical Therapy, University of Florida, Gainesville, FL.
Cardiac dysfunction and respiratory muscle weakness are primary features in patients with early onset Pompe disease. To reduce the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa-/- mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9) or bi-monthly injections of recombinant human GAA (ERT). Cardiac function and morphology was assessed one and three months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at three months post-treatment in all groups. Gaa-/- animals receiving either AAV9 or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9 treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at three months post-injection. Neither AAV9 nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9 animals. Glycogen deposition was significantly elevated in Gaa-/- and ERT but not AAV9 when compared to control animals. These results indicate systemic delivery of either strategy improves cardiac function but improvement in respiratory parameters and glycogen reduction are limited to AAV9 therapy at three-months post treatment in a murine model of Pompe disease.
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
402. Mesenchymal Stromal Cells Can Ameliorate the Progressive Phenotype of Dog With Duchenne Muscular Dystrophy
Yuko N. Kasahara,1,2 Hiromi H. Kinoh,1,2 Mutsuki Kuraoka,1 Tomoko Chiyo,1 Hironori Okada,1,2 Nana Tsumita,1 Kiwamu Imagawa,3 Katsuhiko Tachibana,3 Shin’ichi Takeda,1 Takashi Okada.1,2 1 Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan; 2 Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan; 3JCR Pharmaceuticals Co., Ltd., Ashiya, Kobe, Japan.
Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality that exhibits skeletal muscle weakness with chronic inflammation. Multipotent mesenchymal stromal cells (MSCs) could be a potential therapeutics because of their immunosuppressive properties and ability of differentiation to myogenic cells in situ. In the present study, we examined the strategies for effective cell transplantation to develop a novel approach for functional recovery of the skeletal muscles using a dog model of Duchenne muscular dystrophy. Methods: Human bone marrow-derived MSCs were intravenously injected into two littermates of canine X-linked muscular dystrophy in Japan (CXMD J ) at weekly interval for 8 weeks without immunosuppression. Clinical phenotypes on transplanted dogs were analyzed by using blood exams, physical capacity, MRI and histology compared with two non-injected littermates as controls. Results: The downregulation of inflammation in the lower legs of MSCs-treated CXMDJ was confirmed by magnetic resonance imaging analysis. Immunofluorescence analysis showed decreased area of IgG-positive necrotic myofibers and also increased number of developmental myosin heavy chain-positive regenerative fibers. Impaired tetanic force of gastrocnemius of CXMDJ recovered in hMSCs-treated CXMDJ, from 25% to 80% of wild type. Although CXMDJ showed progressive muscle atrophy in the all four limbs, exercise intolerance and abnormal locomortion, we observed improved phenotypes in the hMSCs-treated CXMDJ along with the improved pace of flip-flop and running. Conclusion: We demonstrated that the systemic injection of MSCs ameliorates the progressive phenotype in CXMDJ. The therapeutic effects might be associated with the production of paracrine or endocrine factors that regulate inflammation, and might also stimulate the proliferation of endogenous stem cells at the injured muscle tissue. This strategy of MSCs treatment would be promising for the future DMD cell therapy.
403. Engineering Micro-Dystrophin AAV Vector for Clinical Translation
Chady H. Hakim,1 Yongping Yue,1 Yi Lai,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, University of Missouri, Columbia, MO.
1
Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy affecting boys. It is caused by the loss of a cellular structural protein called dystrophin. Dystrophin preserves the integrity of the muscle cell membrane by providing a molecular link between the cytoskeleton and the extracellular matrix. The absence of dystrophin destabilizes the muscle membrane and promotes muscle death. AAV micro-dystrophin gene therapy has transformative potentials to significantly ameliorate DMD. Unfortunately, AAV therapy is challenged by destructive cellular immunity to newly expressed dystrophin. To overcome this obstacle, we engineered a double restrictive AAV microgene vector. Specifically, we limited off-target expression in non-muscle tissue with a muscle specific promoter and removed untoward expression in antigen-presenting cells using S159