Recombinant adeno-associated virus vector-based gene transfer for defects in oxidative metabolism

Defects in oxidative metabolism may be caused by mutations either in nuclea r genes or in mitochondrial DNA (mtDNA). We tested the hypothesis that reco mbinant adeno-associated virus (rAAV) could be used to complement mtDNA mut ations. AAV vector constructs were designed to express the reporter gene en coding green fluorescent protein (GFP), fused to a targeting presequence th at directed GFP to be translocated into mitochondria. These vectors mediate d expression of mitochondrial-localized GFP, as indicated by fluorescence m icroscopy and electron microscopy, in respiring human embryonic kidney 293 cells and nonrespiring mtDNA-deficient (rho 0) cells. However, when sequenc es encoding hydrophobic segments of proteins normally encoded by mtDNA were inserted between the presequence and GFP, mitochondrial import failed to o ccur. In similar experiments, a fusion was created between pyruvate dehydro genase (PDH) E1 alpha subunit, a nuclear-encoded mitochondrial gene with it s own targeting presequence, and GFP. With this construct, expression of GF P was observed in mitochondria in vitro and in vivo. We conclude that the h ydrophobicity of mtDNA-encoded proteins limits their ability to be transpor ted from the cytoplasm. However, rAAV-based gene therapy may hold promise f or gene therapy of PDH deficiency, the most common biochemically proven cau se of congenital lactic acidosis.