LEBER HEREDITARY OPTIC NEUROPATHY: GENE THERAPY

描述(由申请人提供)：在前一个赠款周期中，我们在确定LHON的病因和测试LHON的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，尽管复合体I活性的轻微下降似乎不足以诱发疾病。由于目前还没有将DNA直接导入线粒体的技术，我们克服了这一缺陷，构建了线粒体基因的“核版本”，然后通过在阅读框架上附加靶向序列(异位表达)，将细胞质合成的蛋白质靶向线粒体。当被送到含有突变ND4的培养细胞中时，呼吸功能恢复了。这对患者的治疗也有帮助吗？为了帮助回答这个问题，我们现在建议在一个与LHON非常接近的动物模型系统中测试我们的假设，即氧化磷酸化和/或活性氧物种(ROS)的减少会导致视神经病变。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。 (1)我们将通过异位表达人工合成的错义基因来测试突变的ND4蛋白的隐性负面效应，该基因导致ND4蛋白中340位氨基酸的精氨酸替代组氨酸，与线粒体DNA中G11778A突变产生的组氨酸替代相同。我们将异体感染含有正常线粒体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)我们将研究核酶的能力，旨在减少与LHON最常见的复杂I亚单位(ND4)的表达，从而对细胞功能产生不利影响。我们将通过将核酶与RNA导入信号偶联来将核酶导入线粒体，然后通过氧化磷酸化、ROS、细胞生长和凋亡的检测来评估体外的细胞病变效应。 (3)检测ND4核酶和异位表达的突变型ND4基因诱导视神经变性的能力。我们将它们单独或一起传递到小鼠的视觉系统中(使用同时表达核酶和突变基因的载体)，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尸检测量来分析损伤的机制、程度和损伤部位。

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.