Objective: To demonstrate the high efficiency and rapidity of allotopic expression of a normal human ND4 subunit of complex I in the vertebrate retina using a self-complementary adeno-associated virus (scAAV) vector for ocular gene delivery to treat acute visual loss in Leber hereditary optic neuropathy (LHON). Methods: The nuclear-encoded human ND4 subunit fused to the P1 isoform of subunit C of adenosine triphosphate synthase (ATPc) mitochondrial targeting sequence and FLAG epitope was packaged in scAAV2 capsids or single-stranded (ss) AAV2 capsids. These constructs were injected into the vitreous cavities of mice. The contralateral eyes were injected with scAAV-green fluorescent protein (GFP). One week later, pattern electroretinograms and gene expression of the human ND4 subunit and GFP were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed relative to Thy1.2-labeled retinal ganglion cells (RGCs). Results: Pattern electroretinogram amplitudes remained normal in eyes inoculated with scAAV-ND4FLAG, ssAAV-ND4FLAG, and GFP. Confocal microscopy revealed the typical perinuclear mitochondrial expression of scAAV-ND4FLAG in almost the entire retinal flat mount. In contrast, scAAV-GFP expression was cytoplasmic and nuclear. Relative to Thy1.2-positive RGCs, quantification of scAAV-ND4FLAG-positive RGCs was 91% and that of ssAAV-ND4FLAG-positive RGCs was 51%. Conclusion: Treatment of acute visual loss due to LHON may be possible with a normal human ND4 subunit gene of complex I, mutated in most cases of LHON, when delivered by an scAAV vector. Clinical Relevance: Unlike most retinal degenerations that result in slowly progressive loss of vision over many years, LHON due to mutated mitochondrial DNA results in apoplectic, bilateral severe and usually irreversible visual loss. For rescue of acute visual loss in LHON, a highly efficient and rapid gene expression system is required.
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