Mutations in mitochondrial DNA lead to a spectrum of neurodegenerative diseases for which no effective treatment exists. The most common of these is Leber's hereditary optic neuropathy (LHON) caused by mutations in subunit genes (ND1, ND4 or ND6) of NADH dehydrogenase, which is complex I of the respiratory chain. This application builds on the successes of the current grant that pioneered an adeno-associated virus (AAV) vector to which a mitochondrial targeting sequence (MTS) was appended to the viral envelope. The modified vector delivered the NADH dehydrogenase subunit 4 (ND4) gene directly to the mitochondria for prevention of visual loss in mice also injected with a mutated G11778A ND4 responsible for half of all LHON cases. We will now design, modify and test the efficacy and safety of a clinically relevant vector for treatment of this mitochondrial disease by delivery of genes encoding the normal human ND1, ND4 and ND6 subunits to affected cells and tissues; for rescue of cultured human LHON cells harboring each of the three mutations causing LHON and also a transgenic mouse we developed by injection of the MTS AAV containing mutant human ND4 into the blastocyst. This mouse has visual loss progressing to blindness a year after birth, optic nerve head swelling followed by atrophy and degeneration of ganglion cells, which are the characteristic hallmarks of LHON patients.
Our Aims are: (1) To facilitate translational studies for LHON by developing MTS AAV vectors to deliver each of the ND1, ND4 and ND6 subunit genes directly to the mitochondria and test expression to rescue respiration in cybrid cells with100% mutated ND1, ND4 or ND6. We also develop a single AAV cassette that accommodates all three genes that would require a single IND for FDA approval. (2) To evaluate biological effects of intravitreal delivery of MTS AAV vectors in normal mice that result in mitochondrial gene transfer without adverse effects.(3) To rescue visual loss and optic nerve degeneration in transgenic LHON mice, we carry out treatment at stages that closely parallel the human disease before and after RGC loss and optic atrophy, using viral titers in LHON transgenic mice that in our current experiments resulted in rescue without adverse effects. We hope to identify the conditions for long-term rescue of optic neuropathy in mice, so that this approach can be tested in a phase I/II clinical designed to restore the vision of our patients with all three of the common LHON mutations.
Mutations in mitochondrial DNA lead to a spectrum of neurodegenerative diseases for which no effective treatment exists. We propose to use the AAV vector that has been safely tested in many human clinical trials, to deliver the affected gene to the mitochondria. The AAV cassette accommodates genes of up to approximately 3,300 nucleotides (self-complementary), thus providing a platform for introduction of almost any mitochondrial gene. Thus, our technology is also applicable to treatment of the myriad of diseases caused by mutated mitochondrial DNA included in the category of orphan diseases affecting approximately 25 million Americans.
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