title: Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy.
abstract: A G to A transition at nucleotide 11778 in the ND4 subunit gene of complex I was the first point mutation in the mitochondrial genome linked to a human disease. It causes Leber Hereditary Optic Neuropathy, a disorder with oxidative phosphorylation deficiency. To overcome this defect, we made a synthetic ND4 subunit compatible with the "universal" genetic code and imported it into mitochondria by adding a mitochondrial targeting sequence. For detection we added a FLAG tag. This gene was inserted in an adeno-associated viral vector. The ND4FLAG protein was imported into the mitochondria of cybrids harboring the G11778A mutation, where it increased their survival rate threefold, under restrictive conditions that forced the cells to rely predominantly on oxidative phosphorylation to produce ATP. Since assays of complex I activity were normal in G11778A cybrids we focused on changes in ATP synthesis using complex I substrates. The G11778A cybrids showed a 60% reduction in the rate of ATP synthesis. Relative to mock-transfected G11778A cybrids, complemented G11778A cybrids showed a threefold increase in ATP synthesis, to a level indistinguishable from that in cybrids containing normal mitochondrial DNA. Restoration of respiration by allotopic expression opens the door for gene therapy of Leber Hereditary Optic Neuropathy.
authors.affiliation: Department of Ophthalmology, Neuro-Opthalmology Service, University of Florida College of Medicine, Gainesville, FL 32610, USA. neurop@att.net
authors.forename: John
authors.initials: J
authors.lastname: Guy
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Qi
authors.forename: Francesco
authors.initials: F
authors.lastname: Pallotti
authors.forename: Eric A
authors.initials: EA
authors.lastname: Schon
authors.forename: Giovanni
authors.initials: G
authors.lastname: Manfredi
authors.forename: Valerio
authors.initials: V
authors.lastname: Carelli
authors.forename: Andrea
authors.initials: A
authors.lastname: Martinuzzi
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.forename: Alfred S
authors.initials: AS
authors.lastname: Lewin
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002470:Cell Survival; D002478:Cells, Cultured; D004272:DNA, Mitochondrial / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D042967:Electron Transport Complex I; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D006822:Hybrid Cells / Q000502:physiology; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D029242:Optic Atrophy, Hereditary, Leber / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D010085:Oxidative Phosphorylation; D017354:Point Mutation; D016324:Sequence Tagged Sites
chemical_list: D004272:DNA, Mitochondrial; D000255:Adenosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I