Molecular Remedy of Mitochondrial Defects

描述（由申请人提供）：本资助申请的总体目标是确定潜在的治疗领域和/或因氧化磷酸化系统功能失调而导致的疾病的补救措施。据报道，许多人类疾病与线粒体质子转运NADH-醌氧化还原酶（复合物I）的缺陷有关。复合物I的缺陷使其功能失调，导致以下三个问题：（1）呼吸链氧化NADH的能力受损;（2）该酶泵送质子的能力受损;（3）活性氧（ROS）的产生。在这3个问题中，与线粒体不能氧化NADH和/或ROS引起的损伤相比，在三个质子易位位点中的任何一个处的质子泵送的损伤似乎不存在严重的健康危害。 面包酵母线粒体缺乏复合物I，但含有单亚基NADH脱氢酶（Ndi 1）。我们构建了携带NDI 1基因的重组腺相关病毒（rAAV-NDI 1）并转导了哺乳动物细胞系。到目前为止，在我们测试的所有细胞中，NDI 1基因都可以表达。表达的Ndi 1在其自身前导序列的引导下被正确导入线粒体，并作为宿主细胞呼吸链的一员。此外，在生长停滞的人类细胞以及分化的啮齿动物多巴胺能神经细胞中观察到Ndi 1的功能表达。这些结果表明，NDI 1基因为复杂I相关疾病的基因治疗提供了一个潜在的有用工具。 计划在赠款期内进行的研究如下。(1)使用rAAV-NDI 1在大鼠和小鼠的各种组织中功能性表达酵母NDI 1基因。(2)构建由复合物I缺陷引起的人类疾病的动物模型。(3)Ndi 1酶保护免受复合物I缺陷引起的组织降解。

DESCRIPTION (provided by applicant): The overall goal of this grant application is to identify potential area of treatment and/or remedies for the diseases that result from dysfunctional oxidative phosphorylation system. It has been reported that many human diseases are associated with defects in the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). Defects in complex I, which render it dysfunctional, result in the following three problems: (1) impaired ability of the respiratory chain to oxidize NADH; (2) impaired ability of this enzyme to pump protons; (3) production of reactive oxygen species (ROS). Of these 3 problems, impairment of proton pumping at any one of the three proton translocation sites does not appear to present a severe health hazard when compared to the inability of mitochondria to oxidize NADH and/or damage caused by ROS. Baker's yeast mitochondria lack complex I but contain instead a single-subunit NADH dehydrogenase (Ndi1). We have constructed a recombinant adeno-associated virus carrying the NDI1 gene (rAAV-NDI1) and transduced mammalian cell lines. In all cells we have tested so far, the NDI1 gene can be expressed. The expressed Ndi1 was correctly imported into mitochondria guided by its own leading sequence and acted as a member of the respiratory chain in host cells. In addition, functional expression of the Ndi1 was observed in growth-arrested human cells as well as in differentiated rodent dopaminergic nerve cells. These results indicate that the NDI1 gene provides a potentially useful tool for gene therapy of complex I associated diseases. The studies planned for the grant period are as follows. (1) Functional expression of the yeast NDI1 gene in various tissues of rats and mice using rAAV-NDI1. (2) Construction of animal models for human diseases caused by complex I defects. (3) Protection by the Ndi1 enzyme against tissue degradation caused by complex I deficiencies.