CELLULAR AND ANIMAL MODELS OF MITOCHONDRIAL DISEASE

近年来，一些线粒体DNA突变已被鉴定为 引起母系遗传病，其中大部分与 在童年早期就患有精神发育迟滞。我们建议重点关注两个方面 这两种疾病都与多肽的mtDNA点突变有关 编码基因：线粒体脑肌病、乳酸酸中毒和 卒中样发作(MELAS)和母亲遗传的Leigh综合征 (MILS)(这与另一种疾病，神经病， 共济失调和视网膜色素变性(NARP)。梅拉斯是由线粒体DNA引起的 细胞色素c氧化酶基因第III亚基第nT-9957位突变； MILS/NARP是由于ATP6亚单位的NT-8993突变所致 合成酶基因。 聚焦于这两个突变，我们建议创造细胞和动物 利用三种相关策略建立线粒体疾病模型：(L) Cybrid模型：我们将转移携带这些基因的患者线粒体 突变到不含内源性mtDNA的人p/o细胞，从而 产生p/o细胞质杂交种(胞质杂交种)。这将使我们能够研究 中性人群中基因分型与表型的关系 核/线粒体背景，有助于阐明发病机制 在这些疾病中，目前尚不清楚。(2)蜂窝模型：WE 将开始解决基因治疗方法的可能性 在这些致命的疾病中，通过重新编码ATPase 6基因(在这种情况下 MILS~NARP)和COX III基因(对于MELAS)包含 通过体外突变获得通用遗传密码，增加一个线粒体 靶向序列，并将该构建物转移到核 基因组。重新编码的野生型基因的表达应该会改善 突变细胞中各自突变的影响；a的表达 突变基因应在野生型中产生显性-负性表型 细胞。我们还将突变细菌ATPase 6和COX Ill，以模拟 在一个更简单、更容易操纵的系统中的无序。(3)动物 模型：将应用相同的核重新编码和重定向概念 创造NARP和MELAS的转基因小鼠模型，这将是 第一个线粒体疾病的动物模型。

In recent years, a number of mtDNA mutations have been identified that cause maternally-inherited diseases, the majority of which are associated with mental retardation in early childhood. We propose to focus on two such disorders, both associated with mtDNA point mutations in polypeptide coding genes: mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), and maternally-inherited Leigh syndrome (MILS) (which is genetically related to another disease, neuropathy, ataxia, and retinitis pigmentosa, or NARP). MELAS is due to a mtDNA mutation at nt-9957 in subunit III of the cytochrome c oxidase gene; MILS/NARP is due to a mutation at nt-8993 in subunit 6 of the ATP synthetase gene. Focusing on these 2 mutations, we propose to create cellular and animal models of mitochondrial disease, using three related strategies: (l) Cybrid models: We will transfer patient mitochondria harboring these mutations to human p/o cells containing no endogenous mtDNA, thereby creating p/o-cytoplasmic hybrids (cybrids). This will enable us to study the relationship between genotype and phenotype in a neutral nuclear/mitochondrial background, and will help clarify the pathogenesis of these disorders, which is obscure at present. (2) Cellular models: We will begin to address the possibility of a genetic approach to treatment of these fatal disorders, by "recoding" the ATPase 6 gene (in the case of MILS~NARP) and COX III gene (in the case of MELAS) to contain the universal genetic code by in vitro mutagenesis, adding a mitochondrial targeting sequence, and transferring this construct to the nuclear genome. Expression of a recoded wild-type gene should ameliorate the effects of the respective mutations in mutant cells; expression of a mutant gene should create a dominant-negative phenotype in wild-type cells. We will also mutate bacterial ATPase 6 and COX Ill to mimic the disorder in a simpler, more-easily-manipulatable system. (3) Animal models: The same nuclear recoding-and-retargeting concept will be applied to create transgenic mouse models of NARP and MELAS, which would be the first animal models of mitochondrial disease.