New Transformation Technology for Gene Editing of Human Mitochondrial DNA Associated With Mitochondrial Diseases

与线粒体疾病相关的人类线粒体 DNA 基因编辑的新转化技术

• 批准号: 10382077
• 负责人: Hajime Sakai
• 金额: $ 22.5万
• 依托单位: NAPIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382077
• 关键词: Affect; Aging; Alzheimer' s Disease; Basic Science; CRISPR/Cas technology; Cell Respiration; Cells; Cellular Structures; Chloroplast DNA; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; DNA; Diabetes Mellitus; Disease; Eye; Functional disorder; Future; Genes; Genetic Diseases; Genome; Goals; Grant; Hereditary Disease; Human; Human Genetics; Human Genome; Kearns-Sayre syndrome; Leber' s Hereditary Optic Neuropathy; Life; MELAS Syndrome; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Muscle; Mutation; Neurodegenerative Disorders; Organ; Organelles; Organism; Parkinson Disease; Patients; Phase; Plants; Plasmids; Predisposition; Process; Production; Reporting; Research; Rice; Site-Directed Mutagenesis; Small Business Innovation Research Grant; Somatic Cell; Technology; Testing; United States National Institutes of Health; Vision; Yeasts; developmental disease; disease-causing mutation; gene gun; gene therapy; genetic information; human disease; in vivo; mitochondrial DNA mutation; novel; plasmid DNA; protein biomarkers; repaired

PROJECT SUMMARY Human mitochondrial DNA (mtDNA) encodes 37 genes in a small genome of 16,569 bp, which accounts for 0.005% of the total human genome size. Despite the small size of this genetic information, mtDNA is indispensable to human cells. As mitochondria are critical for cellular respiration and ATP production, mitochondria are often referred to as “the Powerhouses of the cell”. Because of their importance, mutations in mtDNA can cause severe diseases and disorders in humans. These include but are not limited to muscular/neurodegenerative and developmental disorders such as Kearns-Sayre syndrome (KSS), Leber’s Hereditary Optic Neuropathy (LHON) and MELAS disorders. Mutations in mtDNA are also suggested to correlate with a predisposition for common diseases like diabetes, Alzheimer’s disease, Parkinson’s disease and even aging. Although the causal relationships between certain mutations in mtDNA and their corresponding diseases were reported decades ago, cures have not been realized due to the difficulty in accessing the sub-cellular structures in vivo. Also, CRISPR technology has not been applicable to mitochondria until recently and corresponding gene therapy of mtDNA still remains challenging in humans. The major challenge of mitochondrial gene editing is the lack of DNA transformation approaches for human mitochondria. No selectable marker gene has been developed that enables mitochondrial transformation in human cells. As part of our efforts for mitochondrial transformation in crop plants, we have used a novel selectable marker gene to develop a method that has shown good efficacy in rice and yeast. In this proposed project, we will apply this method to human mitochondria. A successful demonstration will be a major advancement for the application of our organelle gene editing technology to human mitochondrial DNA, which will facilitate future gene therapy treatments for the repair of mtDNA mutations in patients suffering from severe mitochondrial diseases.

项目摘要 人线粒体DNA（mtDNA）在16,569 bp的小基因组中编码37个基因，该基因 占人类总基因组大小的0.005％。尽管这个遗传很小 信息，mtDNA对于人类细胞是必不可少的。因为线粒体对于细胞至关重要 呼吸和ATP产生，线粒体通常被称为“ 细胞”。由于其重要性，mtDNA中的突变会引起严重的疾病，并且 人类的疾病。这些包括但不限于肌肉/神经退行性和 发育障碍，例如Kearns-Sayre综合征（KSS），Leber的遗传性视神经 神经病（LHON）和MELAS疾病。还建议mtDNA中的突变 与糖尿病，阿尔茨海默氏病等常见疾病的易感性相关， 帕金森氏病甚至衰老。尽管某些人之间的因果关系 数十年前报道了mtDNA及其相应疾病的突变，治疗 由于难以在体内访问亚细胞结构，因此未能实现。还， CRISPR技术直到最近才适用于线粒体 mtDNA的基因疗法在人类中仍然受到挑战。 线粒体基因编辑的主要挑战是缺乏DNA转化方法 对于人类线粒体。尚未开发出可选的标记基因 人类细胞中的线粒体转化。作为我们为线粒体努力的一部分 作物植物的转化，我们使用了一种新型的可选标记基因来发展 在大米和酵母中表现出良好效率的方法。在这个建议的项目中，我们将申请 这种方法是人类线粒体的方法。成功的演示将是一个重大进步 为了将我们的细胞器基因编辑技术应用于人的线粒体DNA， 将促进未来的基因治疗治疗以修复患者mtDNA突变 患有严重的线粒体疾病。