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J Guy, A Lewin, X Qi, W Hauswirth; Gene Therapy with the ND4 Subunit Gene Re-coded in the Universal Genetic Code Rescues a Mitochondrial Deficiency Causing Leber Hereditary Optic Neuropathy . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4599.
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Purpose: To rescue a deficiency in oxidative phosphorylation to a cybrid cell line harboring the G11778A LHON mutation in mitochondrial DNA. Methods: Cybrid cell lines created from patient's tissue with the LHON G11778A mitochondrial DNA mutation in a normal nuclear background showed a 60% reduction in the rate of ATP synthesis. To overcome this deficiency, we re-coded the entire ND4 subunit of mtDNA using the universal genetic code, imported the translated protein into the mitochondria with a mitochondrial targeting sequence (allotopic expression) derived from the P1 isoform of subunit c of ATP synthase (ATPc). For detection of import a FLAG epitope tag was added at the C-terminus. The P1ND4F fusion gene was inserted in an adeno-associated viral(AAV) vector and LHON cybrid cells were transfected. Cells were grown in glucose-rich media, then placed in glucose-free medium containing galactose as the main carbon source for glycolysis. This medium forces the cells to rely predominantly on oxidative phosphorylation to produce ATP. Cells harboring complex I mutations have a severe growth defect in galactose medium. Results: FLAG immunocytochemistry revealed a typical punctate mitochondrial pattern that co-localized with the mitochondrion-specific dye MitoTracker Red, suggesting that P1ND4F was localized in mitochondria. Western blot analysis showed the imported P1ND4F fusion protein of a size consistent with that of the imported polypeptide. Cell survival after 3 days in the glucose deficient galactose media was 3-fold greater for the allotopically transfected P1ND4F cybrids than mock-transfected cybrids (p<0.005). Assays of complex I activity in whole cells by the reduction of cytochrome c with NADH revealed no significant reduction in G11778A cybrids compared to wild-type cybrids. Therefore, we focused on changes in ATP synthesis using the complex I substrates malate and pyruvate. Relative to the wild-type cell line, G11778A mutant cybrids showed a 60% reduction in the rate of ATP synthesis. Allotopic expression of ND4 reversed this deficiency in ATP synthesis. Relative to mock-transfected cybrids, G11778A cybrids complemented with P1ND4F showed a 3-fold increase in the rate of ATP synthesis (p=0.02). In fact, this degree of recovery lead to rates of ATP synthesis that were virtually indistinguishable from the corresponding wild-type cell line. Conclusions: Restoration of ATP synthesis to a cell line harboring the LHON G11778A mutation by allotopic expression of a normal ND4 may be the initial step in a gene-based treatment for this blinding disorder
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