Recombinant adeno-associated virus vector-based gene transfer for defects in oxidative metabolism.
Defects in oxidative metabolism may be caused by mutations either in nuclear genes or in mitochondrial DNA (mtDNA). We tested the hypothesis that recombinant adeno-associated virus (rAAV) could be used to complement mtDNA mutations. AAV vector constructs were designed to express the reporter gene encoding green fluorescent protein (GFP), fused to a targeting presequence that directed GFP to be translocated into mitochondria. These vectors mediated expression of mitochondrial-localized GFP, as indicated by fluorescence microscopy and electron microscopy, in respiring human embryonic kidney 293 cells and nonrespiring mtDNA-deficient (rho 0) cells. However, when sequences encoding hydrophobic segments of proteins normally encoded by mtDNA were inserted between the presequence and GFP, mitochondrial import failed to occur. In similar experiments, a fusion was created between pyruvate dehydrogenase (PDH) E1 alpha subunit, a nuclear-encoded mitochondrial gene with its own targeting presequence, and GFP. With this construct, expression of GFP was observed in mitochondria in vitro and in vivo. We conclude that the hydrophobicity of mtDNA-encoded proteins limits their ability to be transported from the cytoplasm. However, rAAV-based gene therapy may hold promise for gene therapy of PDH deficiency, the most common biochemically proven cause of congenital lactic acidosis.
Owen R
I V
IV
Department of Pediatrics, University of Florida, Gainesville, FL 32610, USA.
Lewin
A P
AP
Peel
A
A
Wang
J
J
Guy
J
J
Hauswirth
W W
WW
Stacpoole
P W
PW
Flotte
T R
TR
D000140:Acidosis, Lactic / Q000151:congenital / Q000628:therapy; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases; D008957:Models, Genetic; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000378:metabolism; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D004272:DNA, Mitochondrial; D008164:Luminescent Proteins; C501005:MT-ATP6 protein, human; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D010100:Oxygen