LEBER HEREDITARY OPTIC NEUROPATHY: GENE THERAPY

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DESCRIPTION (provided by applicant): During the previous grant cycle we have made major strides towards determining the pathogenesls and testing a treatment for LHON. First, we discovered that ND4 mutant cells have a severe reduction in ATP synthesis, even though mild reductions in complex I activity appear insufficient to induce disease. Since no technology exists to introduce DNA directly into mitochondria, we overcame this deficiency in oxidative phosphorylation by constructing a "nuclear version" of the mitochondrial gene then targeted the cytoplasmically synthesized protein to the mitochondria by using a targeting sequence appended to the reading frame (allotopic expression). When delivered to cultured cells containing a mutant ND4, respiratory function was restored. Could this also be useful in the treatment of patients? To help answer this we now propose to test our hypothesis that reductions in oxidative phosphorylation and/or reactive oxygen species (ROS) induce optic neuropathy in an animal model system that closely approximates LHON. Our specific aims logically progress from understanding the physiological consequences of a mutated ND4 gene and loss of ND4 expression to creating a novel animal disease model of LHON. (1) We will test for a recessive negative effect of mutated ND4 protein by allotopic expression of a synthetic mlssense gene that results in the same substitution of an arginine for histidine, at amino acid 340, in the ND4 protein as that produced by the G11778A mutation in mitochondrial DNA. We will allotopically infect cells harboring normal mitochondrial DNA, and then evaluate for cytopathic effects by measuring cell growth, apoptosis, respiration and ROS. (2) We will investigate the ability of ribozymes designed to reduce expression of the complex I subunit (ND4) most commonly associated with LHON to adversely impact cellular function. We will import the ribozyme into mitochondria by coupling it with an RNA import signal and then evaluate for cytopathic effects in vitro using assays of oxidative phosphorylation, ROS, cell growth and apoptosis. (3) We will test the ability of the ND4 ribozyme and allotopically expressed mutant ND4 gene to induce optic nerve degeneration. We will deliver them to the mouse visual system individually or together (using a vector expressing both ribozyme and mutant gene), then analyze the mechanism, extent and foci of injury using serial in vivo digital fundus photography, MRI and post-mortem measurements of ganglion cell and axon damage.