232. Short_Term, In Vivo Correction of VLCAD Deficiency in Mice: Steps toward a Gene Therapy for Cardiomyopathy
Authors: Tien Nguyen, Jan Daniels, Lawrence Merritt, Dietrich Matern, Jerry Vockley, Philip A. Wood, David B. Schowalter
Journal: Molecular Therapy (2006)

Cardiomyopathy is an important cause of morbidity and mortality in children. Deficiency of very_long_chain acyl_CoA dehydrogenase (VLCAD), one of several nuclear encoded mitochondrial enzymes that catalyze the initial step in the beta_oxidation of straight chain fatty acids, often presents with cardiomyopathy and/or sudden death. How VLCAD deficiency causes cardiomyopathy is unclear, but appears to be related to the accumulation of toxic long_chain acyl_carnitine species rather than a block in energy metabolism. A recent report of a 5_year_old with VLCAD deficiency note resolution of her hypertrophic cardiomyopathy while on a strict low fat diet supplemented with medium chain triglycerides and carnitine. While dietary therapy for VLCAD deficiency has been beneficial to some, many people to not respond. Intercurrent illness with diminished oral intake, exercise, or fasting all increase demand for fatty acid metabolism and can potentially trigger catastrophic events even while on dietary therapy.The murine knock_out model of VLCAD deficiency has a milder disease than that seen in humans, possibly due to differences in the substrate specificity and/or tissue distribution of VLCAD between humans and mice. Nevertheless, this mouse is the only model of VLCAD deficiency and remains a good model to study the effects of reconstituted VLCAD activity will have on fatty acid metabolism.Here we report here the development of a recombinant AAV (serotype 8) vector that expresses the human VLCAD gene (AAV8_ hVLCAD) using the CMV promoter. In vitro plasmid studies demonstrate a functional, appropriate sized VLCAD protein is produced by the expression cassette. To demonstrate in vivo activity, 3 × 1011 AAV8_hVLCAD vector genomes were administered via the tail vein into VLCAD deficient mice. Two weeks after injection, mice receiving AAV8_hVLCAD showed a reduction in accumulated long chain acyl_carnitine species to a level that is similar to that seen in wild_type litter mates. Western analysis of liver, skeletal muscle, and heart extracts using the novel Pep_1 anti_VLCAD antibody demonstrate hVLCAD expression in the liver, and to a lesser extent in the skeletal muscle and heart. This work demonstrates the ability of human VLCAD to correct the serologic abnormalities seem in the VLCAD deficient mice. Cardiomyopathy is an important cause of morbidity and mortality in children. Deficiency of very_long_chain acyl_CoA dehydrogenase (VLCAD), one of several nuclear encoded mitochondrial enzymes that catalyze the initial step in the beta_oxidation of straight chain fatty acids, often presents with cardiomyopathy and/or sudden death. How VLCAD deficiency causes cardiomyopathy is unclear, but appears to be related to the accumulation of toxic long_chain acyl_carnitine species rather than a block in energy metabolism. A recent report of a 5_year_old with VLCAD deficiency note resolution of her hypertrophic cardiomyopathy while on a strict low fat diet supplemented with medium chain triglycerides and carnitine. While dietary therapy for VLCAD deficiency has been beneficial to some, many people to not respond. Intercurrent illness with diminished oral intake, exercise, or fasting all increase demand for fatty acid metabolism and can potentially trigger catastrophic events even while on dietary therapy. The murine knock_out model of VLCAD deficiency has a milder disease than that seen in humans, possibly due to differences in the substrate specificity and/or tissue distribution of VLCAD between humans and mice. Nevertheless, this mouse is the only model of VLCAD deficiency and remains a good model to study the effects of reconstituted VLCAD activity will have on fatty acid metabolism. Here we report here the development of a recombinant AAV (serotype 8) vector that expresses the human VLCAD gene (AAV8_ hVLCAD) using the CMV promoter. In vitro plasmid studies demonstrate a functional, appropriate sized VLCAD protein is produced by the expression cassette. To demonstrate in vivo activity, 3 × 1011 AAV8_hVLCAD vector genomes were administered via the tail vein into VLCAD deficient mice. Two weeks after injection, mice receiving AAV8_hVLCAD showed a reduction in accumulated long chain acyl_carnitine species to a level that is similar to that seen in wild_type litter mates. Western analysis of liver, skeletal muscle, and heart extracts using the novel Pep_1 anti_VLCAD antibody demonstrate hVLCAD expression in the liver, and to a lesser extent in the skeletal muscle and heart. This work demonstrates the ability of human VLCAD to correct the serologic abnormalities seem in the VLCAD deficient mice.