C9orf72 frontotemporal dementia (FTD) and amyotrophic lateral sclerosis(ALS): using patient cells and CRISPR to reveal therapeutic approaches

项目总结/摘要 在C9 orf 72基因的单个等位基因中杂合六核苷酸（GGGGCC）重复扩增是最常见的。 额颞叶痴呆（FTD）和肌萎缩侧索硬化症（ALS）的常见已知遗传原因， 致命和不可逆的神经退行性疾病。鉴于FTD没有有效的治疗方法（ 阿尔茨海默氏症相关痴呆）和ALS，新的治疗策略是迫切需要的。针对 通过CRISPR/Cas9基因编辑的C9 orf 72基因本身可能提供治疗性干预。但我们需要 了解C9 orf 72基因的生物学，以便采用基因编辑策略。 这项工作提出了CRISPR基因编辑技术的新应用，以编辑或沉默致病基因。 FTD/ALS患者来源的iPSC中的C9 orf 72疾病等位基因。随着这些目标的实现，我们将 系统地评价了三种互补的方法，用于沉默一种有害的重复扩增， C9 orf 72基因：（1）仅携带重复扩增的非编码DNA的双等位基因切除（Aim 1），（2）等位基因- 特异性切除含有重复扩增的突变等位基因（Aim 1），（3）调节区破坏 选择性沉默C9 orf 72重复扩增（目的3）。我们将检查这些编辑的能力 在与疾病相关的细胞类型中纠正疾病病理学的策略-人类皮层和运动神经元。 我们已经开发出快速和强大的方法来产生神经元从人类诱导多能干细胞 （iPSC）来源于对照和患者。对编辑的对照细胞系的分析将使我们能够筛选 精确的基因编辑对正常细胞功能和健康产生意想不到的影响。我们的发现不仅 促进我们对潜在治疗方法的理解，但也将告知我们对C9 orf 72的理解 生物学，包括C9 orf 72基因调控和疾病的潜在机制。这和我们未来的研究 将开发一个管道，系统地评估编辑策略，是潜在的治疗。

PROJECT SUMMARY/ABSTRACT A heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene is the most frequent known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal and irreversible neurodegenerative diseases. Given that there are no effective treatments for FTD (an Alzheimer’s-related dementia) and ALS, novel therapeutic strategies are urgently needed. Targeting the C9orf72 gene itself by CRISPR/Cas9 gene editing may provide a curative intervention. However, we need to learn about the biology of the C9orf72 gene in order to employ gene editing strategies. This work proposes novel applications of CRISPR gene editing technology to edit or silence the pathogenic C9orf72 disease allele in FTD/ALS patient derived iPSC. With the completion of these aims, we will have systematically evaluated three complementary methods for silencing a deleterious repeat expansion in the C9orf72 gene: (1) bi-allelic excision of non-coding DNA harboring only the repeat expansion (Aim 1), (2) allele- specific excision of the mutant allele containing the repeat expansion (Aim 1), (3) regulatory region disruption to selectively silence the C9orf72 repeat expansion (Aim 3). We will examine the ability of these editing strategies to correct disease pathology in cell types relevant to disease – human cortical and motor neurons. We have developed fast and robust methods to generate neurons from human induced pluripotent stem cells (iPSCs) derived from controls and patients. Analysis of edited control cell lines will allow us to screen for unanticipated effects of precise gene edits on normal cellular function and fitness. Our findings will not only advance our understanding of potential therapeutic approaches, but will also inform our understanding C9orf72 biology, including C9orf72 gene regulation and potential mechanisms of disease. This and our future studies will develop a pipeline for systematically evaluating editing strategies that are potentially curative.