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175. Double-Layered Cell Sheets Transplantation That Achieves Durable Factor VIII Delivery in the Mouse Model of Hemophilia A
Diabetes, Metabolic and Genetic Diseases I 175. Double-Layered Cell Sheets Transplantation That Achieves Durable Factor VIII Delivery in the Mouse Model of Hemophilia A
Hideto Matsui,1 Masahi Noda,1 Rie Utoh,2 Midori Shima,3 Teruo Okano,2 Mitsuhiko Sugimoto.1 1 Regulatory Medicine for Thrombosis, Nara Medical University, Kashihara, Nara, Japan; 2Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan; 3Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
Hemophilia A is an inherited bleeding disorder caused by a deficiency of the procoagulant cofactor VIII (FVIII). Currently, the patients with hemophilia A are being treated through repeated intravenous injection of FVIII concentrates. Since FVIII is a secreted protein and the tight regulation of its expression is not necessary, ex vivo gene therapy strategies for hemophilia A might be good for the therapeutic alternative. We previously demonstrated that therapeutic levels of plasma FVIII were documented from hemophilia A mice over 300 days, in which lentivirally-engineered blood outgrowth endothelial cells (BOECs) sheet were implanted subcutaneously (Tatsumi K et.al. PLoS One 2013 8(12):e83280). To enhance the viability of the implanted BOECs which boost the FVIII expression, double-layered cell sheets transplantation was performed by placing the recovered BOECs sheet onto the fibroblast sheet directly. FVIII transduced BOECs and non-transduced fibroblasts were cultured on temperature-responsive culture dish independently. Both cells were recovered as a contiguous cell sheet. Subcutaneous co-transplantation studies of BOEC and fibroblast sheet indicated that all hemophilia A mice treated with cyclophosphamide expressed between 40-50 mU/mL levels of FVIII without developing anti-FVIII inhibitory antibodies over 200 days. In contrast, the hemophilia A mice without any prior immuno-suppressive treatment developed neutralizing antiFVIII antibodies between 5-10 Bethesda units. These therapeutic FVIII levels were two times higher than the group of hemophilia A mice that were transplanted BOECs sheet alone. Histological examination in the part of the mice revealed that the combination of BOEC and fibroblast sheet was structured as flat clusters without any cells infiltration. In conclusion, tissue engineering approach using double-layered cell sheets are viable for persistent tissue survival and providing therapeutic values. This novel ex vivo gene transfer strategy can provide the safe and efficacious delivery of FVIII in hemophilia A and merits further assessment.
176. An Internally Tagged Methylmalonyl-CoA Mutase (Mut) Displays In Vivo Activity after AAV9 Mediated Gene Delivery
Jessica Schneller,1,2 Julien Senac,2 Randy Chandler,2 Charles P. Venditti.2 1 Biomedical Engineering, SUNY Stony Brook, Stony Brook, NY; 2 Organic Acid Research Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD. Introduction: Methylmalonic acidemia (MMA) is an autosomal recessive metabolic disorder most frequently resulting from mutations in the mitochondrial localized enzyme, methylmalonyl-CoA mutase (MUT). A mouse model replicating the most severe form of MMA in which there is complete loss of Mut expression (Mut-/-) displays neonatal lethality but has been successfully used to demonstrate proof-of-principle for gene therapy using AAV vectors. To model the hepato-, renal and cerebral manifestations of MMA, we have generated several lines of transgenic/knock-out mice to create viable animal models of MMA. Mut-/-; TgMckMut mice express wild type Mut in the skeletal muscle under the control of the murine creatine kinase S70
promoter and display severe biochemical perturbations. A milder form of Mut deficiency is modeled by mice that constitutively express the mouse orthologue of a well-characterized partial activity mutation (Mut-/-; TgMutG715V). We sought to develop a functional, tagged Mut enzyme that could be used to examine efficacy and transgene expression in the varied mouse models of MMA. Methods: The 5’ end of the Mut gene was replaced with a synthetic sequence that contained the endogenous mitochondrial importation sequence, a mitochondrial protease cleavage site, and a V5 tag and was used to create AAV9-Chicken Beta Actin (CBA)-V5-Mut. AAV9-CBA-V5-Mut (1e11 GC) was systemically delivered to adult Mut-/-; TgMckMut (n=4) or Mut-/-; TgMutG715V (n=2) mice. Following gene therapy, weight, metabolites, 1-C-13 propionate oxidation and/ or expression of the V5-tagged Mut enzyme were assayed in treated mice. Results: Mut-/-; TgMckMut mice demonstrated a significant weight increase over a fifteen day time period to levels comparable to untreated heterozygous littermates. Plasma methylmalonic acid concentrations decreased from ~250µM to ~100µM (p<0.01), and ability to oxidize propionate increased. In liver extracts from treated Mut-/-; TgMutG715V mice but not controls, detection of a V5 tagged enzyme of the predicted size of a Mut-V5 fusion was apparent 1 week after injection. Conclusions: We have created a new gene therapy vector that expresses the Mut enzyme internally tagged with a V5 epitope. Studies performed with Mut-/-; TgMckMut mice demonstrate the V5-Mut fusion enzyme has full biological activity in vivo. The facile detection of the V5 tag in Mut-/-; TgMutG715V mice shows that V5-Mut can be assayed when co-expressed with a missense mutation representing the most common class of mutations seen in humans with MUT MMA. Vectors designed to express V5-Mut should be useful to assay activity and biodistribution after AAV gene therapy in varied models of MMA.
177. Recapitulating the Triglyceride-Lowering Effect of Cold Exposure By Overexpression of Fibroblast Growth Factor-21 Gene
Mingming Gao,1 Yongjie Ma,1 Kexiu Song,1 Hao Sun,1 Dexi Liu.1 Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA.
1
Cold exposure reduces blood triglyceride level via repressing lipid synthesis and enhancing lipid oxidation, providing a potential option for hyperlipidemia therapy. However, cold exposure is not a practical therapeutic approach in humans due to its unacceptable requirements. As the effect of cold exposure is associated with elevated expression of fibroblast growth factor-21 (Fgf21) in the liver and brown adipose tissue (BAT), we assessed in this study whether the metabolic benefits of cold exposure in lowering blood triglyceride can be recapitulated by overexpressing Fgf21. Overexpression of Fgf21 was achieved by hydrodynamic delivery of plasmid driven by an albumin promoter for Fgf21 expression. Our results show that hydrodynamic transfer of the Fgf21 plasmid exclusively elevated Fgf21 expression in the liver in a dose-dependent manner. Fgf21 overexpression greatly improves triglyceride metabolism in regular as well as high-fat diet-induced obese mice, as evidenced by improved profiles of oral fat tolerance tests and reduced fasting triglyceride levels in blood. Mechanistically, improvements in triglyceride metabolism mediated by Fgf21 transfer were achieved by repressed lipid synthesis in the liver and enhanced lipid oxidation in BAT. At the molecular level, Fgf21 transfer repressed transcription of a variety of pivotal genes involved in lipid synthesis in liver, including Srebp1c, Fas, Scd1, Ppargamma2 and Mgat1, while increasing mRNA levels of Cpt1-alpha and Acadl, suggesting that the hepatic lipid synthesis was repressed while oxidation enhanced. In fact, transfer of the Fgf21 remained effective in attenuating hyperlipidemia in mice fed with 30% fructose Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
Hideto Matsui,1 Masahi Noda,1 Rie Utoh,2 Midori Shima,3 Teruo Okano,2 Mitsuhiko Sugimoto.1 1 Regulatory Medicine for Thrombosis, Nara Medical University, Kashihara, Nara, Japan; 2Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan; 3Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
Hemophilia A is an inherited bleeding disorder caused by a deficiency of the procoagulant cofactor VIII (FVIII). Currently, the patients with hemophilia A are being treated through repeated intravenous injection of FVIII concentrates. Since FVIII is a secreted protein and the tight regulation of its expression is not necessary, ex vivo gene therapy strategies for hemophilia A might be good for the therapeutic alternative. We previously demonstrated that therapeutic levels of plasma FVIII were documented from hemophilia A mice over 300 days, in which lentivirally-engineered blood outgrowth endothelial cells (BOECs) sheet were implanted subcutaneously (Tatsumi K et.al. PLoS One 2013 8(12):e83280). To enhance the viability of the implanted BOECs which boost the FVIII expression, double-layered cell sheets transplantation was performed by placing the recovered BOECs sheet onto the fibroblast sheet directly. FVIII transduced BOECs and non-transduced fibroblasts were cultured on temperature-responsive culture dish independently. Both cells were recovered as a contiguous cell sheet. Subcutaneous co-transplantation studies of BOEC and fibroblast sheet indicated that all hemophilia A mice treated with cyclophosphamide expressed between 40-50 mU/mL levels of FVIII without developing anti-FVIII inhibitory antibodies over 200 days. In contrast, the hemophilia A mice without any prior immuno-suppressive treatment developed neutralizing antiFVIII antibodies between 5-10 Bethesda units. These therapeutic FVIII levels were two times higher than the group of hemophilia A mice that were transplanted BOECs sheet alone. Histological examination in the part of the mice revealed that the combination of BOEC and fibroblast sheet was structured as flat clusters without any cells infiltration. In conclusion, tissue engineering approach using double-layered cell sheets are viable for persistent tissue survival and providing therapeutic values. This novel ex vivo gene transfer strategy can provide the safe and efficacious delivery of FVIII in hemophilia A and merits further assessment.
176. An Internally Tagged Methylmalonyl-CoA Mutase (Mut) Displays In Vivo Activity after AAV9 Mediated Gene Delivery
Jessica Schneller,1,2 Julien Senac,2 Randy Chandler,2 Charles P. Venditti.2 1 Biomedical Engineering, SUNY Stony Brook, Stony Brook, NY; 2 Organic Acid Research Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD. Introduction: Methylmalonic acidemia (MMA) is an autosomal recessive metabolic disorder most frequently resulting from mutations in the mitochondrial localized enzyme, methylmalonyl-CoA mutase (MUT). A mouse model replicating the most severe form of MMA in which there is complete loss of Mut expression (Mut-/-) displays neonatal lethality but has been successfully used to demonstrate proof-of-principle for gene therapy using AAV vectors. To model the hepato-, renal and cerebral manifestations of MMA, we have generated several lines of transgenic/knock-out mice to create viable animal models of MMA. Mut-/-; TgMckMut mice express wild type Mut in the skeletal muscle under the control of the murine creatine kinase S70
promoter and display severe biochemical perturbations. A milder form of Mut deficiency is modeled by mice that constitutively express the mouse orthologue of a well-characterized partial activity mutation (Mut-/-; TgMutG715V). We sought to develop a functional, tagged Mut enzyme that could be used to examine efficacy and transgene expression in the varied mouse models of MMA. Methods: The 5’ end of the Mut gene was replaced with a synthetic sequence that contained the endogenous mitochondrial importation sequence, a mitochondrial protease cleavage site, and a V5 tag and was used to create AAV9-Chicken Beta Actin (CBA)-V5-Mut. AAV9-CBA-V5-Mut (1e11 GC) was systemically delivered to adult Mut-/-; TgMckMut (n=4) or Mut-/-; TgMutG715V (n=2) mice. Following gene therapy, weight, metabolites, 1-C-13 propionate oxidation and/ or expression of the V5-tagged Mut enzyme were assayed in treated mice. Results: Mut-/-; TgMckMut mice demonstrated a significant weight increase over a fifteen day time period to levels comparable to untreated heterozygous littermates. Plasma methylmalonic acid concentrations decreased from ~250µM to ~100µM (p<0.01), and ability to oxidize propionate increased. In liver extracts from treated Mut-/-; TgMutG715V mice but not controls, detection of a V5 tagged enzyme of the predicted size of a Mut-V5 fusion was apparent 1 week after injection. Conclusions: We have created a new gene therapy vector that expresses the Mut enzyme internally tagged with a V5 epitope. Studies performed with Mut-/-; TgMckMut mice demonstrate the V5-Mut fusion enzyme has full biological activity in vivo. The facile detection of the V5 tag in Mut-/-; TgMutG715V mice shows that V5-Mut can be assayed when co-expressed with a missense mutation representing the most common class of mutations seen in humans with MUT MMA. Vectors designed to express V5-Mut should be useful to assay activity and biodistribution after AAV gene therapy in varied models of MMA.
177. Recapitulating the Triglyceride-Lowering Effect of Cold Exposure By Overexpression of Fibroblast Growth Factor-21 Gene
Mingming Gao,1 Yongjie Ma,1 Kexiu Song,1 Hao Sun,1 Dexi Liu.1 Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA.
1
Cold exposure reduces blood triglyceride level via repressing lipid synthesis and enhancing lipid oxidation, providing a potential option for hyperlipidemia therapy. However, cold exposure is not a practical therapeutic approach in humans due to its unacceptable requirements. As the effect of cold exposure is associated with elevated expression of fibroblast growth factor-21 (Fgf21) in the liver and brown adipose tissue (BAT), we assessed in this study whether the metabolic benefits of cold exposure in lowering blood triglyceride can be recapitulated by overexpressing Fgf21. Overexpression of Fgf21 was achieved by hydrodynamic delivery of plasmid driven by an albumin promoter for Fgf21 expression. Our results show that hydrodynamic transfer of the Fgf21 plasmid exclusively elevated Fgf21 expression in the liver in a dose-dependent manner. Fgf21 overexpression greatly improves triglyceride metabolism in regular as well as high-fat diet-induced obese mice, as evidenced by improved profiles of oral fat tolerance tests and reduced fasting triglyceride levels in blood. Mechanistically, improvements in triglyceride metabolism mediated by Fgf21 transfer were achieved by repressed lipid synthesis in the liver and enhanced lipid oxidation in BAT. At the molecular level, Fgf21 transfer repressed transcription of a variety of pivotal genes involved in lipid synthesis in liver, including Srebp1c, Fas, Scd1, Ppargamma2 and Mgat1, while increasing mRNA levels of Cpt1-alpha and Acadl, suggesting that the hepatic lipid synthesis was repressed while oxidation enhanced. In fact, transfer of the Fgf21 remained effective in attenuating hyperlipidemia in mice fed with 30% fructose Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy