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J. Guy, R. D. Koilkonda, V. Porciatti, W. W. Hauswirth, H. Yu; Gene Therapy for Mitochondrial Disease. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2525.
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© ARVO (1962-2015); The Authors (2016-present)
To develop gene therapy for mitochondrial disease. Mutations in mitochondrial DNA lead to a spectrum of neurodegenerative diseases for which no treatment exists. The most common is Leber Hereditary Optic Neuropathy caused by a mutated ND4 subunit of complex I.
To direct the AAV virion to the mitochondria, a targeting sequence (COX8) linked to GFP was be appended to AAV capsid protein VP2. The human ND4 gene with a FLAG epitope under the control of a mitochondrial promoter was packaged as a single-stranded (ss) or double-stranded (sc) AAV vector. Translocation to mitochondria was assessed by testing for wild-type ND4 gene, RT-PCR of ND4FLAG mRNA, immunocytochemistry and immunoblotting for FLAG. The ability of mitochondrially targeted AAV-ND4 gene therapy to rescue the defective respiration of LHON cells was gauged by cell growth and respiration. In vivo studies included intraocular injection of the mito-targeted AAV into the vitreous of normal mice. Safety of human ND4 gene expression in mice was monitored by pattern electroretinograms (PERG).
Mitochondria isolated from transfected LHON cells revealed a 1.4Kb band in addition to endogenous mtDNA. RT-PCR revealed ND4FLAG mRNA. ATP synthesis increased 12 fold. Expression of ND4FLAG with single-stranded AAV took 7 weeks in the mouse retina, due to the time required for generation of a complementary ND4 DNA strand. In contrast expression with the double-stranded scAAV-ND4 occurred within a week and co-localized with mitochondria labeled by a porin antibody and within Thy1.2 positive retinal neurons. At one or two months respectively, 75% or 98% of the retinal ganglion cells expressed human ND4FLAG. PERG amplitudes remained normal.
The novel technology developed here may deliver virtually any mitochondrial gene, and thus may provide the platform to treat not only visual loss but also the myriad of disabilities experienced by patients with diseases caused by mutated mitochondrial DNA.
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