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

父项目摘要/摘要 C9orf72的单个等位基因中的六核苷酸(GGGGCC)重复扩展是导致 额颞叶痴呆(FTD)和肌萎缩侧索硬化症(ALS)，其中两种致命的神经退行性变 目前还没有治愈的方法。由于FTD(一种与阿尔茨海默氏症相关的痴呆症)没有有效的治疗方法 而ALS，迫切需要新的治疗方法。CRISPR/Cas9基因编辑靶向C9orf72 提出了一种很有前途的候选疗法。提高我们对C9orf72生物学的理解将有助于 使用基因编辑策略。这项工作利用CRISPR的新应用程序来专门编辑或 沉默FTD/ALS患者来源的诱导多能干细胞(IPSCs)中的患病等位基因。正在完成 这些目标对C9-FTD/ALS的三种互补基因编辑策略进行了系统评估： 1)含有重复扩增的第一内含子内的双等位基因切除(目标1)，(2)等位基因特异性切除 突变的等位基因包含重复扩大(目标2)，(3)选择性地扰乱调节区 停止C9orf72重复扩展(目标3)。此外，我们还比较了编辑策略的能力 纠正与疾病相关的细胞类型--人类大脑皮层和运动神经元--的疾病病理 通过快速和稳健的方法，我们开发了从对照和 病人。对对照细胞系编辑结果的分析使我们能够筛选精确的基因编辑和 评估对正常细胞功能和健康的意外影响。我们的发现不仅将推动我们的 寻找潜在的治疗方法，但也有助于我们对C9orf72生物学的理解 生物学，包括C9orf72基因是如何调控的，以及疾病的潜在机制。这个和我们的 未来的研究将开发一条管道，系统地评估潜在的新颖编辑策略 治疗C9-FTD/ALS。我的工作将发展父母基金的目标3(目标1)，并进一步扩展它(目标2)

PARENT PROJECT SUMMARY/ABSTRACT A hexanucleotide (GGGGCC) repeat expansion in a single allele of C9orf72 is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal neurodegenerative of which there is currently no cure. Since there are no effective treatments for FTD (an Alzheimer’s related dementia) and ALS, there is a critical need for novel therapeutics. Targeting C9orf72 with CRISPR/Cas9 gene editing presents a promising candidate therapy. Improving our understanding of the biology of C9orf72 will facilitate employing gene editing strategies. This work utilizes novel applications of CRISPR to specifically edit or silence the diseased allele in FTD/ALS patient-derived induced pluripotent stem cells (iPSCs). Completing these aims generates a systematic evaluation of three complementary gene editing strategies for C9-FTD/ALS: 1) bi-allelic excision within the first intron harboring the repeat expansion (Aim 1), (2) allele-specific excision of the mutant allele harboring the repeat expansion (Aim 2), (3) disruption of regulatory regions to selectively silence theC9orf72 repeat expansion (Aim 3). Furthermore, we compare the ability of the editing strategies to correct disease pathology in cell types relevant to disease–human cortical and motor neurons–made possible by the fast and robust methods we developed to generate neurons from iPSCs derived from controls and patients. Analysis of the editing outcomes in control cell lines enables us to screen for precise gene editing and evaluate un anticipated effects on normal cellular function and fitness. Our findings will not only advance our search for potential therapeutic approaches, but also inform our understanding of the biology of C9orf72 biology, including how the C9orf72 gene is regulated and the mechanisms underlying disease. This and our future studies will develop a pipeline for systematically evaluating novel editing strategies that are potentially curative for C9-FTD/ALS. My work will develop Aim 3 of the parent grant (Aim 1) and expand it further (Aim 2)