LEBER HEREDITARY OPTIC NEUROPATHY: GENE THERAPY

描述（由申请人提供）：在上一个拨款周期中，我们在确定LHON的发病机制和测试治疗方法方面取得了重大进展。首先，我们发现ND4突变细胞的ATP合成严重减少，尽管复合物I活性的轻微减少似乎不足以诱导疾病。由于没有将DNA直接引入线粒体的技术，我们通过构建线粒体基因的“核版本”来克服氧化磷酸化的缺陷，然后通过附加在阅读框上的靶向序列（同种异体表达）将细胞质合成的蛋白质靶向到线粒体。当传递到含有ND4突变体的培养细胞时，呼吸功能恢复。这对治疗病人也有用吗？为了帮助回答这个问题，我们现在提议在一个非常接近LHON的动物模型系统中验证我们的假设，即氧化磷酸化和/或活性氧（ROS）的减少会诱导视神经病变。我们的具体目标从理解ND4基因突变和ND4表达缺失的生理后果到创建一种新的LHON动物疾病模型。

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.