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

项目摘要/摘要 C9orf72基因单等位基因GGGGCC重复扩增次数最多 额颞部痴呆(FTD)和肌萎缩侧索硬化症(ALS)常见的已知遗传原因，两个 致命和不可逆转的神经退行性疾病。鉴于没有有效的治疗FTD(AND)的方法 阿尔茨海默氏症(阿尔茨海默病相关的痴呆)和肌萎缩侧索硬化症，迫切需要新的治疗策略。目标是 通过CRISPR/Cas9基因编辑，C9ORF72基因本身可能提供一种治疗性干预。然而，我们需要 了解C9orf72基因的生物学知识，以便使用基因编辑策略。 这项工作提出了CRISPR基因编辑技术在编辑或沉默致病病毒方面的新应用 FTD/ALS患者发生IPSC的C9ORF72等位基因随着这些目标的实现，我们将拥有 系统地评估了三种互补的方法来压制有害的重复扩张 C9ORF72基因：(1)仅含有重复扩增的非编码DNA的双等位基因切除(目标1)，(2)等位基因- 含有重复扩增的突变等位基因的特异性切除(目标1)，(3)调控区中断 选择性沉默C9orf72重复扩增(目标3)。我们将检查这些编辑的能力 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的策略。 我们已经开发出快速而强大的方法来从人类诱导的多能干细胞中产生神经元 (IPSCs)来源于对照组和患者。对编辑的控制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的意想不到的影响。我们的发现不仅会 增进了我们对潜在治疗方法的理解，但也将有助于我们对C9orf72的理解 生物学，包括C9orf72基因调控和潜在的疾病机制。这和我们未来的研究 将开发一条管道，对可能治愈的编辑策略进行系统评估。

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.