Purpose: To evaluate the long-term effects of mitochondrial gene transfer of mutant human NADH ubiquinone oxidoreductase subunit VI (hND6T14484C) in the mouse eye. Methods: Adult mice were injected intravitreally with mitochondrial-targeted adeno-associated virus carrying either hND6T14484C or mitochondrial encoded mCherry. The delivery and expression of the interest gene were detected by polymerase chain reaction (PCR), quantitative PCR (qPCR), and immunostaining. The pathologic effects of the mutant gene in live mice were assessed with RNA-seq, serial spectral domain optical coherence tomography (SD-OCT), and pattern electroretinogram (PERG). Results: Delivered hND6 was found 30-fold greater than endogenous mouse ND6 in microdissected retinal ganglion cells of hND6-injected mice. Compared to controls injected with mCherry, PERG amplitude of hND6 mice dropped significantly at 3 (P = 0.0023), 6 (P = 0.0058), and 15 (P = 0.031) months after injection. SD-OCT revealed swelling of the optic nerve head followed by the progressive retinal and optic nerve atrophy in hND6 mice. Furthermore, RNA-seq data showed a change in 381 transcripts’ expression in these mice compared to mCherry mice. Postmortem analysis showed hND6 mice had marked atrophy of the entire optic nerve, from the globe to the optic chiasm, and a significant loss of retinal ganglion cells compared to age-matched control mice (P = 1.7E-9). Conclusions: Delivered hND6T14484C induces visual loss and optic neuropathy in mice, the hallmarks of human Leber's hereditary optic neuropathy (LHON). Translational Relevance: Results from this study will help establish a novel strategy not only to generate an LHON animal model but also to provide a potential to treat this or any other mitochondrial diseases.

Introduction: Therapies for Leber hereditary optic neuropathy (LHON), in common with all disorders caused by mutated mitochondrial DNA, are inadequate. We have developed two gene therapy strategies for the disease: mitochondrial-targeted and allotopic expressed and compared them in a mouse model of LHON.Methods: A LHON mouse model was generated by intravitreal injection of a mitochondrialtargeted Adeno-associated virus (AAV) carrying mutant human NADH dehydrogenase 4 gene (hND4/m.11778G>A) to induce retinal ganglion cell (RGC) degeneration and axon loss, the hallmark of the human disease. We then attempted to rescue those mice using a second intravitreal injection of either mitochondrial-targeted or allotopic expressed wildtype human ND4. The rescue of RGCs and their axons were assessed using serial pattern electroretinogram (PERG) and transmission electron microscopy.Results: Compared to non-rescued LHON controls where PERG amplitude was much reduced, both strategies significantly preserved PERG amplitude over 15 months. However, the rescue effect was more marked with mitochondrial-targeted therapy than with allotopic therapy (p = 0.0128). Post-mortem analysis showed that mitochondrial-targeted human ND4 better preserved small axons that are preferentially lost in human LHON.Conclusions: These results in a pre-clinical mouse model of LHON suggest that mitochondrially-targeted AAV gene therapy, compared to allotopic AAV gene therapy, is more efficient in rescuing the LHON phenotype.