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R. D. Koilkonda, T.-H. Chou, V. Porciatti, W. W. Hauswirth, J. Guy; Self-Complementary AAV Induces Rapid and Highly Efficient Allotopic Expression of the Human ND4 Complex I Subunit in the Mouse Visual System. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4494.
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© ARVO (1962-2015); The Authors (2016-present)
Lebers Hereditary Optic Neuropathy (LHON), caused by mutated mitochondrial DNA results in apoplectic bilateral severe and usually irreversible visual loss. For rescue of visual loss in LHON, a highly efficient and rapid gene expression system is required. The present study is to demonstrate the high efficiency, rapidity and safety of allotopic expression of a normal human ND4 subunit of complex I in ganglion cells (RGCs) of the vertebrate retina using the self-complementary AAV vector for ocular gene delivery
The nuclear encoded human ND4 subunit fused to the ATPc mitochondrial targeting sequence and FLAG epitope was packaged in double-stranded self-complementary (sc) AAV2 capsids or single-stranded (ss) AAV2 capsids. These constructs were injected into the vitreous cavity of mice. The contralateral eyes were injected with scAAV-GFP. One week later, gene expression of the human ND4 subunit and GFP were evaluated by performing immunofluorescence microscopy. Quantitative analysis of ND4FLAG injected eyes was assessed relative to Thy1.2 labeled RGCs. Pattern electroretinogram (PERG) and flashelectroretinogram (FERG) were recorded to address the safety issue upon gene injections.
Confocal microscopy revealed the typical perinuclear mitochondrial expression of scAAV-ND4FLAG in almost the entire retinal flat mounts. In contrast, sc-AAV-GFP expression was cytoplasmic and nuclear. Relative to Thy1.2-positive RGCs, quantification of scAAV-ND4FLAG-positive RGCs was 91% and ssAAV-ND4FLAG-positive RGCs was 52%. PERG and FERG analysis showed no significant difference in the average amplitudes and latencies one week after the viral injection in the scAAV-ND4FLAG, ssAAV-ND4 FLAG and GFP inoculated eyes (p>0.05), indicating no difference in the functional status of the retina.
Treatment of acute visual loss from LHON may be possible with a normal human ND4 subunit of complex I, mutated in most cases of LHON, when delivered by a double-stranded AAV vector.
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