Transgenic Mitomice Models for Treatment of Blinding Diseases

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DESCRIPTION (provided by applicant): While mutated mitochondrial (mt)DNA is firmly linked to the blindness of Leber's hereditary optic neuropathy (LHON), a bona fide animal model with mutated mtDNA complex I subunits that would enable probing the pathogenesis of the optic neuropathy and testing of potential avenues for therapy has yet to be developed. It had not been possible to produce animal models of human diseases caused by mitochondrial mutations due to the barrier in delivering DNA into mitochondria. We circumvented this barrier by appending to the adenoassociated virus (AAV) a mitochondrial targeting sequence (MTS) to the viral protein capsid to direct the mutant human ND4 gene (responsible for half of all LHON cases) into murine mitochondria. When driven by a human mitochondrial promoter, expression of mutant human ND4 in murine mitochondria induced visual loss with a progressive demise of ganglion cells in the retina and their axons comprising the optic nerve, thus recapitulating the hallmarks of the human LHON disorder. Our motivation in pursuing this project is to develop gene therapy for untreatable mitochondrial diseases that lead to blindness. For this, we have to (1) develop a plausible therapy, an MTS AAV to deliver normal genes to affected mitochondria and (2) create animal models with mutated complex I subunit genes that recapitulate the human disorder in which to test potential treatments and the pathogenesis of optic neuropathy at stages that resemble the human disease. To generate a transgenic mouse we delivered the MTS AAV containing the mutant human ND4 allele to embryonic stem cells by microinjection into the blastocyst of the mouse, generating offspring with progressive retinal ganglion cell (RGC) demise and optic neuropathy with pattern electroretinogram (PERG) amplitudes declining steadily to noise levels one year after birth. To monitor mitochondrial gene expression in live animals we added a mitochondrial encoded red fluorescent protein (mCherry) to the MTS AAV construct containing mutant human ND4 that could be visualized by laser scanning ophthalmoscopy (LSO) in live offspring and so far in three generations of their progeny. Our Specific Aims logically progress from understanding the consequences of mutant NADH dehydrogenase subunit ND4 in mice to developing additional transgenic lines expressing the two other mutant human complex I subunits (ND1 and ND6) responsible for LHON.