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

C9orf72 额颞叶痴呆 (FTD) 和肌萎缩侧索硬化症 (ALS)：利用患者细胞和 CRISPR 揭示治疗方法

• 批准号: 10590420
• 负责人: Bruce R Conklin
• 金额: $ 23.93万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10590420
• 关键词: ALS patients; Alleles; Alzheimer' s Disease; Alzheimer' s disease related dementia; Amyotrophic Lateral Sclerosis; Biology; C9ORF72; CRISPR/Cas technology; Cell Line; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Dementia; Disease; Evaluation; Excision; Frontotemporal Dementia; Future; Genes; Genetic; Goals; Induced pluripotent stem cell derived neurons; Introns; Link; Mendelian disorder; Methods; Motor Neurons; Nerve Degeneration; Neurodegenerative Disorders; Nucleic Acid Regulatory Sequences; Outcome; Pathology; Patients; Therapeutic; Work; cell type; effective therapy; fitness; frontotemporal lobar dementia-amyotrophic lateral sclerosis; human disease; improved; induced pluripotent stem cell; mutant; novel; novel therapeutics; parent grant; parent project; therapeutic gene

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)

父项目摘要/摘要 C9orf72 的单个等位基因中的六核苷酸 (GGGGCC) 重复扩增是最常见的原因 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疾病 目前尚无治愈方法，因为 FTD（一种阿尔茨海默病相关痴呆症）尚无有效治疗方法。 和 ALS 方面，迫切需要通过 CRISPR/Cas9 基因编辑来靶向 C9orf72 的新疗法。 提高我们对 C9orf72 生物学的理解将有助于 这项工作利用 CRISPR 的新颖应用来特异性编辑或 沉默 FTD/ALS 患者来源的诱导多能干细胞 (iPSC) 中的患病等位基因。 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) 包含重复扩增的第一个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 突变等位基因包含重复扩增（目标 2），(3) 选择性破坏调控区域 沉默 C9orf72 重复扩展（目标 3） 此外，我们比较了编辑策略的能力。 与疾病相关的细胞类型（人类皮质和运动神经元）的正确疾病病理学成为可能 通过我们开发的快速而稳健的方法，从对照和衍生的 iPSC 中生成神经元 分析对照细胞系的编辑结果使我们能够筛选精确的基因编辑和治疗。 评估对正常细胞功能和健康的意外影响我们的发现不仅会推进我们的研究。 寻找潜在的治疗方法，同时也让我们了解 C9orf72 的生物学 生物学，包括 C9orf72 基因的调控方式以及疾病的机制。 未来的研究将开发一个系统评估潜在的新颖编辑策略的管道 我的工作将制定家长补助金的目标 3（目标 1）并进一步扩展它（目标 2）。