Genetic Correction of Maternally Inherited Diseases

母体遗传疾病的基因矫正

• 批准号: 6784130
• 负责人: MIN-XIN GUAN
• 金额: $ 17.58万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6784130
• 关键词: aminoacid tRNA ligase; cell line; complementary DNA; congenital disorders; electrophoresis; functional /structural genomics; gene expression; gene therapy; hybrid cells; mitochondrial DNA; molecular pathology; point mutation; protein biosynthesis; technology /technique development; transfection; transfection /expression vector; transfer RNA; translation factor

DESCRIPTION (provided by applicant): The objective of this proposed research is to define the role of mitochondrial leucyl-tRNA synthetase and elongation factor TU (EFTU) in the pathogenesis of mutations in the tRNALeu (UUR) that are commonly related to human diseases. This second goal of this study is to explore a way to therapeutically intervene for maternally inherited disease by over-expression of these two proteins in the cybrids carrying pathogenic mutations in the tRNA Leu (UUR). A variety of mitochondrial DNA (mtDNA) mutation have been found to be associated with many clinical abnormalities, including neuromuscular disorders. Of the 100 pathogenic point mutations in mtDNA, >70 occur in tRNA genes. Of these, the most common mutation is the A to G transition at position 3243 (A3243G) in the tRNALeu (UUR) gene, which causes mitochondrial encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS) and other disorders. Mitochondria with this mutated tRNA exhibited a reduced ATP production, which results from quantitative deficiencies in mitochondrial protein synthesis. We hypothesize that the over-expression of human mitochondrial leucyl-tRNA synthetase and EFTU in the disease cell model of the A3243G mutation in the tRNALeu (UUR) gene will correct the mitochondrial translational defects, consequently increasing the level of ATP production. This application proposes two specific aims: 1). Construction of the stably transfected cell lines through transferring human mitochondrial leucyl-tRNA synthetase and EFTU cDNAs into the cybrid cell lines carrying the A3243G mutation and wild type mtDNA. 2). These stably transfected cell lines will be evaluated for the correction of mitochondrial dysfunction by using biochemical and metabolic assays. Success of this protection will define the role of the mitochondrial leucyl-tRNA synthetase and EFTU in the pathogenesis of mutations in the tRNALeu(UUR) gene associated with human diseases. This should provide new insights into the molecular mechanism of maternally inherited disorders. In particular, success in the aim of correcting the biochemical defect associated with A3243G mutation in the tRNA Leu (UUR) gene by overexpressing mitochondrial leucyl-tRNA synthetase, will open the way to therapeutic interventions for maternally inherited diseases. In the long term, fundamental experimental approaches and knowledge, which results from this proposed work would be applicable to many other tRNA gene mutations, associated diseases in humans.

描述（由申请人提供）：这项拟议的研究的目的是定义线粒体亮氨酸-TRNA合成酶和伸长因子TU（EFTU）在trnaleu（UUR）突变的发病机理中通常与人类疾病相关的作用。这项研究的第二个目标是探索一种通过过表达在cybrid中的过表达来探索一种治疗方法，从而探索了tRNA LEU（UUR）中的这两种蛋白质。 已经发现各种线粒体DNA（mtDNA）突变与包括神经肌肉疾病在内的许多临床异常有关。在mtDNA中的100个致病点突变中，> 70发生在tRNA基因中。其中，最常见的突变是在Trnaleu（Uur）基因的3243（A3243G）处的A至G转变，该基因会导致线粒体脑膜病，乳酸酸中毒，中风样症状（Melas）和其他疾病。线粒体使用该突变的tRNA表现出降低的ATP产生，这是由于线粒体蛋白质合成的定量缺陷所致。 我们假设在trnaleu（UUR）基因的A3243G突变的疾病细胞模型中，人线粒体亮氨酸-TRNA合成酶和EFTU的过表达将纠正线粒体翻译缺陷，从而增加ATP产生水平。该应用程序提出了两个具体目标：1）。通过将人线粒体亮氨叶tRNA合成酶和EFTU cDNA转移到带有A3243G突变和野生型mtDNA的Cybrid细胞系中，构建稳定转染的细胞系。 2）。这些稳定的转染细胞系将通过使用生化和代谢测定法进行评估，以校正线粒体功能障碍。 该保护的成功将定义线粒体亮氨酸-TRNA合成酶和EFTU在与人类疾病相关的trnaleu（UUR）基因突变的发病机理中的作用。这应该为母体遗传疾病的分子机制提供新的见解。特别是，在纠正与A3243G突变相关的生物化学缺陷（UUR）基因中相关的生物化学缺陷，通过过表达的线粒体leucyl-tRNA合成酶，将为母遗传疾病的治疗干预措施开辟道路。从长远来看，由这项拟议的工作产生的基本实验方法和知识将适用于许多其他tRNA基因突变，即人类的相关疾病。