Epigenetic Editing of Mutant C9orf72

 描述（由申请人提供）：肌萎缩侧索硬化症（ALS）和额颞叶变性（FTD）是两种相关的神经退行性疾病，具有重叠的临床、病理和遗传特征。ALS/FTD疾病谱是一致致命的，既没有治疗也没有治愈。在这些疾病中加剧或减轻疾病进展的内源性机制尚不清楚。然而，已发现C9 orf 72基因内的突变是ALS/FTD最常见的遗传原因。该突变由六核苷酸重复扩增组成，已提出该六核苷酸重复扩增导致有毒RNA和蛋白质种类的积累。C9 orf 72突变还与突变携带者亚组中的C9 orf 72启动子高甲基化相关。启动子超甲基化似乎可以防止许多与C9 orf 72突变相关的分子畸变，包括DNA重复不稳定性，毒性RNA积累，二肽重复蛋白积累和细胞对应激的脆弱性。C9 orf 72甲基化还可预测携带C9 orf 72突变的FTD患者的病程延长、灰质维持和记忆功能保留。基于这些发现，该提议的假设是突变体C9 orf 72的表观遗传编辑可以调节疾病发病机制。为了验证这一假设，我开发了一种在内源基因组内引入或去除CpG甲基化的新方法，并提出了三个具体目标：（1）确定靶向表观遗传编辑的分子机制和特异性，（2）在患者来源的iPS细胞中引入C9 orf 72超甲基化作为概念验证研究，以表明表观遗传靶向可以是治疗性的，和（3）通过在具有大C9 orf 72重复扩增的iPS细胞中使C9 orf 72启动子去甲基化来开发改进的疾病模型。这些研究将使表观遗传编辑成为现实， 调节神经退行性疾病表型，并将突出一种广泛适用于许多学科的新型表观遗传编辑技术的实用性。

 DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) are two related neurodegenerative diseases which share overlapping clinical, pathologic and genetic features. The ALS/FTD spectrum of diseases is uniformly fatal, and there is neither treatment nor cure. The endogenous mechanisms which exacerbate or mitigate disease progression in these diseases are not clearly understood. However, a mutation within the C9orf72 gene has been discovered as the most common genetic cause of ALS/FTD. The mutation consists of a hexanucleotide repeat expansion which has been proposed to lead to the accumulation of toxic RNA and protein species. C9orf72 mutations are also associated with C9orf72 promoter hypermethylation in a subset of mutation carriers. Promoter hypermethylation appears to protect against many of the molecular aberrations associated with the C9orf72 mutation including DNA repeat instability, toxic RNA accumulation, dipeptide repeat protein accumulation and cellular vulnerability to stress. C9orf72 methylation also predicts prolonged disease duration, maintenance of grey matter, and preservation of memory function in FTD patients with the C9orf72 mutation. Based on these findings, the hypothesis of this proposal is that epigenetic editing of mutant C9orf72 can modulate disease pathogenesis. To test this hypothesis, I have developed a novel method of introducing or removing CpG methylation within the endogenous genome, and propose three specific aims to (1) determine the molecular mechanisms and specificity of targeted epigenetic editing, (2) introduce C9orf72 hypermethylation in patient-derived iPS cells as a proof-of-concept study to show that epigenetic targeting can be therapeutic, and (3) develop improved models of disease by demethylating the C9orf72 promoter in iPS cells with large C9orf72 repeat expansions. These studies will bring to reality the possibility of epigenetic editing as a means of modulating neurodegenerative disease phenotypes, and will highlight the utility of a novel epigenetic editing technique that is broadly applicable across many disciplines.