CELLULAR AND ANIMAL MODELS OF MITOCHONDRIAL DISEASE

线粒体疾病的细胞和动物模型

• 批准号: 6272313
• 负责人: ERIC A. SCHON
• 金额: $ 23.62万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272313
• 关键词: RNase protection assay; adenosinetriphosphatase; ataxia; cell fusion; cellular pathology; central nervous system disorders; disease /disorder model; enzyme activity; gene mutation; genetic disorder; genetic manipulation; genetically modified animals; genotype; human subject; hybrid cells; laboratory mouse; lactic acidosis; mental retardation; mitochondrial DNA; neuromuscular disorder; northern blottings; phenotype; point mutation; posttranscriptional RNA processing; retinitis pigmentosa

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.

近年来，一些线粒体DNA突变已被确定， 导致母体遗传性疾病，其中大多数与 患有智力迟钝的儿童。我们建议把重点放在两个方面 这类疾病都与多肽中的mtDNA点突变有关， 编码基因：线粒体脑肌病，乳酸酸中毒， 中风样发作（MELAS）和母系遗传的Leigh综合征 （MILS）（与另一种疾病，神经病， 共济失调和色素性视网膜炎或NARP）。MELAS是由于线粒体DNA 细胞色素c氧化酶基因的亚基III中nt-9957处的突变; MILS/NARP是由于ATP的亚基6中的nt-8993突变引起的。 合成酶基因 针对这两种突变，我们提出了细胞和动物的基因突变。 线粒体疾病模型，使用三种相关策略：（l） Cybrid模型：我们将转移携带这些细胞的患者线粒体， 突变到不含内源性mtDNA的人p/o细胞，从而 产生p/o-胞质杂种（胞质杂种）。这将使我们能够研究 中性粒细胞中基因型和表型之间关系 核/线粒体背景，并将有助于阐明发病机制 目前尚不清楚(2)细胞模型：我们 将开始探讨基因治疗的可能性 在这些致命的疾病中，通过“重新编码”ATP酶6基因（在这种情况下， MILS~NARP）和考克斯III基因（在MELAS的情况下），以包含 通过体外诱变的通用遗传密码， 靶向序列，并将该构建体转移到细胞核中， 基因组重新编码的野生型基因的表达应该改善 突变细胞中相应突变的影响; 突变基因应在野生型中产生显性阴性表型 细胞我们还将突变细菌ATP酶6和考克斯III，以模拟 在一个更简单，更容易操作的系统中。(3)动物 型号：将采用相同的核重新编码和重新瞄准概念 创建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。