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)

PANEL项目总结/摘要 C9 orf 72的单个等位基因中的六核苷酸（GGGGCC）重复扩增是C9 orf 72的最常见原因。 额颞叶痴呆（FTD）和肌萎缩侧索硬化（ALS），其中两种致命的神经退行性疾病， 目前还没有治愈的方法。由于没有有效的治疗FTD（阿尔茨海默氏症相关的痴呆症） 和ALS，迫切需要新的治疗方法。CRISPR/Cas9基因编辑靶向C9 orf 72 提出了一种很有前途的候选疗法。提高我们对C9 orf 72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的新应用来特异性地编辑或 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。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价： 1)在第一个内含子内进行双等位基因切除，携带重复扩增（Aim 1），（2）等位基因特异性切除 携带重复扩增的突变等位基因（目的2），（3）调节区的破坏， 沉默C9 orf 72重复扩增（目的3）。此外，我们比较了编辑策略的能力， 在与疾病相关的细胞类型-人类皮质和运动神经元-中纠正疾病病理学成为可能 通过我们开发的快速和强大的方法，从来自对照的iPSC中产生神经元， 患者对对照细胞系中编辑结果的分析使我们能够筛选精确的基因编辑， 评估对正常细胞功能和健康的意外影响。我们的发现不仅会促进我们的 寻找潜在的治疗方法，但也告知我们对C9 orf 72生物学的理解 生物学，包括C9 orf 72基因是如何调节的，以及疾病的潜在机制。这和我们 未来的研究将开发一个管道，系统地评估新的编辑策略， 治疗C9-FTD/ALS。我的工作将发展母基金的目标3（目标1），并进一步扩大它（目标2）。