Gene therapy for pyruvate dehydrogenase E1alpha deficiency using recombinant adeno-associated virus 2 (rAAV2) vectors.
To determine the feasibility of gene transfer to correct defects in the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex (PDC), we constructed rAAV vectors that expressed PDH E1alpha, either alone or with a green fluorescent protein tag, from a hybrid cytomegalovirus (CMV) enhancer/chicken beta-actin (CB) promoter. These vectors were functional in vitro, as judged by increased expression of mRNA in vector-transduced deficient cell lines and correction of the biochemical defect in PDH activity in these cells. Approximately 30% of wild-type levels of PDH activity were restored under conditions with which only about 15% of cells were transduced. These same vectors were then used in vivo to transduce neurons within the rat striatum. Gene transfer, expression, and translocation into mitochondria were observed, without any obvious untoward effects. In vivo vector-mediated PDH expression persisted for at least 1 year after injection, indicating the stability of gene transfer. These studies provide the basis for future efforts to develop a recombinant AAV (rAAV)-based gene therapy approach for the correction of PDC deficiency.
Owen
Renius
R
Department of Pediatrics, The General Clinical Research Center, University of Florida, Gainesville, Florida 32610, USA.
Mandel
Ronald J
RJ
Ammini
Chandramohan V
CV
Conlon
Thomas J
TJ
Kerr
Douglas S
DS
Stacpoole
Peter W
PW
Flotte
Terence R
TR
D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line / Q000187:drug effects; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000172:deficiency / Q000235:genetics*; D014161:Transduction, Genetic
D030481:Pyruvate Dehydrogenase (Lipoamide); C065640:pyruvate dehydrogenase E1alpha subunit