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

项目摘要 人类线粒体DNA（mtDNA）编码37个基因，在一个16,569 bp的小基因组中， 占人类基因组总长度的0.005%。尽管这种基因的体积很小， 线粒体DNA是人类细胞不可缺少的。由于线粒体对细胞的 由于线粒体参与呼吸和ATP的产生，线粒体通常被称为“生命的动力库”。 细胞”。由于它们的重要性，mtDNA的突变可能导致严重的疾病， 人类的疾病。这些包括但不限于肌肉/神经退行性疾病， 发育障碍，如Kearns-Sayre综合征（KSS）、Leber遗传性视神经 神经病（LHON）和MELAS疾病。线粒体DNA的突变也被认为 与常见疾病如糖尿病、阿尔茨海默氏症、 帕金森病甚至衰老。虽然某些因果关系 几十年前就报道了线粒体DNA突变及其相应的疾病， 由于难以进入体内亚细胞结构，还有， CRISPR技术直到最近才适用于线粒体， mtDNA的基因治疗在人类中仍然具有挑战性。 线粒体基因编辑的主要挑战是缺乏DNA转化方法 for human人mitochondria线粒体.还没有开发出可选择的标记基因， 人类细胞中的线粒体转化。作为我们研究线粒体的一部分 在农作物转化中，我们使用了一种新的选择标记基因， 该方法在大米和酵母中显示出良好的效果。在这个项目中，我们将 这种方法应用于人类线粒体。成功的演示将是一个重大的进步 将我们的细胞器基因编辑技术应用于人类线粒体DNA， 将促进未来的基因治疗治疗，用于修复患者的mtDNA突变， 患有严重的线粒体疾病

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.