437. Developing a Mouse Model of the Mitochondrial NARP Syndrome Using AAV-Ribozymes for ATP6

Abstract

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly diagnosed inborn error of metabolism. Clinical presentation ranges from mild hypotonia and hypoglycemia in children to sudden unexpected death. Current therapies including avoidance of fasting, vitamin supplementation, and dietary management are effective, but may not prevent acute clinical decompensations associated with infectious illnesses. Together, this makes MCAD deficiency a good model disease for a safe and durable gene delivery system. The adeno-associated virus subtype 2 (AAV) is known to be a durable gene delivery system which can transduce non-dividing cells and has minimal toxicity in phase 1 human clinical trials. We report here the development of a recombinant AAV vector that expresses the human MCAD gene (AAV-hMCAD). Preliminary studies with the expression cassette demonstrated localization of the MCAD protein to the mitochondria, and correction of the metabolic block in fibroblasts isolated from MCAD deficient patients. Intact AAV-hMCAD particles were produced by triple plasmid transfection in 293 cells then characterized by electron microscopy and ELISA assay. Real-time PCR quantified viral genome containing particles. Human MCAD transcript was identified after AAV-hMCAD infection of MCAD deficient human and murine fibroblasts by RT-PCR. Acyl-carnitine analysis revealed functional correction of the metabolic block seen in MCAD deficient fibroblasts after infection with AAV-hMCAD. Together, these data provide important preliminary data for future in vivo studies in MCAD deficient mice.