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J. Guy, X. Qi, A. S. Lewin, W. W. Hauswirth; Mitochondrial Delivery of the Human ND4 Gene Rescues Cultured LHON Cells and Is Expressed in the Mouse Optic Nerve. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4415.
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© ARVO (1962-2015); The Authors (2016-present)
To deliver a gene to the mitochondrion.
To target the AAV vector to the mitochondria we inserted GFP cDNA under the direction of a mitochondrial targeting sequence (MTS) into a modified AAV capsid. We modified this vector by adding the COX8 MTS inserted into the VP2 capsid of AAV at unique EAGI sites. We then linked a normal mitochondrial encoded ND4 subunit gene with an appended FLAG epitope to a mitochondrial promoter, the H strand promoter in an AAV backbone (pTR-UF) containing the inverted terminal repeats. This was delivered to LHON cybrids homoplasmic for the G11778A mutation in mitochondrial DNA. ND4FLAG expression was detected with an anti-FLAG antibody. We also isolated mtDNA from the transfected cybrids using PCR primers flanking the H strand promoter and the 3'mitoND4. To determine if import led to functional complementation, LHON cells were infected, then tested for rescue by measuring cell growth in glucose-deficient galactose media. We tested for rescue of ATP synthesis using the complex I substrates malate and pyruvate as substrates for oxidative phosphorylation. We also injected the mito-targeted AAV into the vitreous of four DBA/1J mice then tested for expression of FLAG.
We found the translocation of viral capsid particles expressing GFP to mitochondria, by co-localization with the mitochondrial specific dye MitoTracker. In addition, the A20 specific antibody co-localized with COX8MTS-GFP suggested import of the fully assembled virion. ND4FLAG expression was detected in the infected cells. Agarose gel electrophoresis of the isolated mtDNA of transfected LHON cybrids revealed the 1.3 Kb band that contained both SfNaI sites in wild-type ND4. A day after transfection and 2 days of growth in galactose we found a 19% increase in LHON cell survival (p <0.05) relative to controls, cybrid cells infected with the same ND4 gene but in a virus without the MTS attached to the GFP expressing VP2. We found a 48% increase in the rate of ATP synthesis in LHON cybrids infected with the mito-targeted AAV expressing wild-type ND4 relative to control (p <0.05). Numerous FLAG positive cells visualized by anti-FLAG-cy3 were seen in the RGC layer of a retinal flat mount seven weeks after mito-targeted AAV injection. Silver enhanced anti-mitoND4FLAG immunogold was seen in optic nerve mitochondria.
Mitochondrial gene delivery may offer hope for the treatment of patients with diseases caused by mutated mitochondrial DNA and aging.
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