title: In vitro characterization and in vivo expression of human very-long chain acyl-CoA dehydrogenase.
abstract: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of fatty acid beta-oxidation that can present at any age with cardiomyopathy, rhabdomyolysis, hepatic dysfunction, and/or nonketotic hypoglycemia. Through the expansion of newborn screening programs an increasing number of individuals with VLCAD deficiency are being identified prior to the onset of symptoms allowing early initiation of therapy. The development of a safe, durable, and effective VLCAD gene delivery system for use at the time of diagnosis could result in a significant improvement in the quality and duration of life for patients with VLCAD deficiency. To this end, we developed a construct containing the human VLCAD cDNA under the control of the strong CMV promoter (pCMV-hVLCAD). A novel rabbit polyclonal anti-VLCAD antibody was prepared using a 24 amino-acid peptide unique to the human VLCAD protein to study human VLCAD expression in immune competent mice. Antibody specificity was demonstrated in Western blots of human VLCAD deficient fibroblasts and in pCMV-hVLCAD transiently transfected VLCAD deficient fibroblasts. Transfected fibroblasts showed correction of the metabolic block as demonstrated by normalization of C14- and C16-acylcarnitine species in cell culture media and restoration of VLCAD activity in cells. Following tail vein injection of pCMV-hVLCAD into mice, we demonstrated expression of hVLCAD in liver. Altogether, these steps are important in the development of a durable gene therapy for VLCAD deficiency.
authors.affiliation: Department of Medical Genetics, Mayo Clinic College of Medicine, Rochester, MN, USA.
authors.forename: J Lawrence
authors.initials: JL
authors.lastname: Merritt
authors.forename: Dietrich
authors.initials: D
authors.lastname: Matern
authors.forename: Jerry
authors.initials: J
authors.lastname: Vockley
authors.forename: Jan
authors.initials: J
authors.lastname: Daniels
authors.forename: Tien V
authors.initials: TV
authors.lastname: Nguyen
authors.forename: David B
authors.initials: DB
authors.lastname: Schowalter
mesh_terms: D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D006801:Humans; D047468:Immunoprecipitation; D008099:Liver / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D044942:Acyl-CoA Dehydrogenase, Long-Chain