177 recapitulating the triglyceride lowering effect of cold exposure by overexpression of fibroblast growth factor 21 gene

177 Recapitulating the Triglyceride Lowering Effect of Cold Exposure By Overexpression of Fibroblast Growth Factor 21 Gene Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American[.]

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 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 Biomedical Engineering, SUNY Stony Brook, Stony Brook, NY; 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