TY - JOUR
T1 - In vitro characterization and in vivo expression of human very-long chain acyl-CoA dehydrogenase
AU - Merritt, J. Lawrence
AU - Matern, Dietrich
AU - Vockley, Jerry
AU - Daniels, Jan
AU - Nguyen, Tien V.
AU - Schowalter, David B.
N1 - Funding Information:
This study was supported by funding from the American Heart Association (0234938N) and Mayo Clinic College of Medicine clinical research funds. Jerry Vockley was supported in part by PHS NIH Grant RO1 DK45482. We also greatly appreciate the generous gift of the anti-VLCAD antibody by Dr. Arnold Strauss (Vanderbilt University Medical Center, Nashville, TN), and hVLCAD cDNA plasmid from Dr. Brage Andresen (Research Unit for Molecular Medicine, University of Aarhus, Aarhus, Denmark). We thank Bambi Anderson for technical assistance.
PY - 2006/8
Y1 - 2006/8
N2 - 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.
AB - 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.
KW - Gene therapy
KW - Hydrodynamic plasmid injection
KW - In vitro correction
KW - VLCAD antibody
KW - Very-long chain acyl-CoA dehydrogenase
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U2 - 10.1016/j.ymgme.2006.02.010
DO - 10.1016/j.ymgme.2006.02.010
M3 - Article
C2 - 16621643
AN - SCOPUS:33746313507
SN - 1096-7192
VL - 88
SP - 351
EP - 358
JO - Molecular genetics and metabolism
JF - Molecular genetics and metabolism
IS - 4
ER -