LEBER HEREDITARY OPTIC NEUROPATHY: GENE THERAPY

描述（由申请人提供）：在上一个资助周期中，我们在确定LHON的病因和测试治疗方面取得了重大进展。首先，我们发现ND 4突变细胞ATP合成严重减少，即使复合物I活性的轻度减少似乎不足以诱导疾病。由于不存在将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”，然后通过使用附加到阅读框的靶向序列将细胞质合成的蛋白质靶向线粒体（异位表达），克服了氧化磷酸化的这种缺陷。当递送到含有突变ND 4的培养细胞时，呼吸功能恢复。这对病人的治疗也有帮助吗？为了帮助回答这个问题，我们现在提出测试我们的假设，即氧化磷酸化和/或活性氧（ROS）的减少诱导视神经病变的动物模型系统，非常接近LHON。我们的具体目标从理解突变的ND 4基因的生理后果和ND 4表达的丧失到创建LHON的新型动物疾病模型，这是合乎逻辑的。 (1)我们将通过合成的有义基因的异位表达来测试突变ND 4蛋白的隐性负效应，该基因导致ND 4蛋白中第340位氨基酸处的精氨酸取代为组氨酸，与G11778 A突变产生的相同。线粒体DNA。我们将异位感染携带正常线粒体DNA的细胞，然后通过测量细胞生长、凋亡、呼吸和ROS来评估细胞病变效应。 (2)我们将研究设计用于减少最常与LHON相关的复合物I亚基（ND 4）表达的核酶对细胞功能产生不利影响的能力。我们将通过将核酶与RNA导入信号偶联将其导入线粒体，然后使用氧化磷酸化、ROS、细胞生长和凋亡测定法评估体外细胞病变效应。 (3)我们将测试ND 4核酶和异位表达的突变ND 4基因诱导视神经变性的能力。我们将它们单独或一起（使用同时表达核酶和突变基因的载体）递送到小鼠视觉系统，然后使用一系列活体数字眼底照相、MRI和神经节细胞和轴突损伤的死后测量来分析损伤的机制、程度和病灶。

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.