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: 10186371
• 负责人: Bruce R Conklin
• 金额: $ 212.63万
• 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/10186371
• 关键词: ALS patients; Address; Alleles; Alzheimer' s Disease; Alzheimer' s disease related dementia; Amyotrophic Lateral Sclerosis; Biological Assay; Biology; C9ORF72; CRISPR/Cas technology; Cell Line; Cell physiology; Cells; Chromosome 13; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA; Dementia; Disease; Engineering; Enhancers; Excision; Exons; Frontotemporal Dementia; Functional disorder; Future; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Enhancer Element; Genetic Polymorphism; Genome; Genomic Segment; Goals; Human; Induced pluripotent stem cell derived neurons; Knock-out; Learning; Link; Mendelian disorder; Methods; Mitotic; Motor Neurons; Mutagenesis; Mutation; Neurodegenerative Disorders; Neuronal Dysfunction; Neurons; Nucleic Acid Regulatory Sequences; Nucleotides; Pathogenicity; Pathology; Patients; Phenotype; Regulation; Regulatory Element; Safety; Series; Site; System; Technology; Testing; Therapeutic; Therapeutic Uses; Time; Untranslated RNA; Work; cell type; cellular pathology; curative treatments; disease phenotype; effective therapy; experimental study; fitness; frontotemporal lobar dementia-amyotrophic lateral sclerosis; gain of function; human disease; in vivo; induced pluripotent stem cell; loss of function; mutant; novel; novel therapeutic intervention; novel therapeutics; preservation; promoter; risk variant; stem cell model; therapeutic gene; therapeutic genome editing; therapy design

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.

项目概要/摘要 C9orf72 基因的单个等位基因中的杂合六核苷酸 (GGGGCC) 重复扩展是最常见的 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，两种 鉴于 FTD（一种致命的、不可逆转的神经退行性疾病）尚无有效的治疗方法。 阿尔茨海默病相关的痴呆）和肌萎缩侧索硬化症（ALS），迫切需要针对这些疾病的新治疗策略。 C9orf72基因本身通过CRISPR/Cas9基因编辑可能提供治疗性干预，但是，我们需要这样做。 了解 C9orf72 基因的生物学，以便采用基因编辑策略。 这项工作提出了 CRISPR 基因编辑技术在编辑或沉默致病基因方面的新应用。 FTD/ALS 患者衍生的 iPSC 中的 C9orf72 疾病等位基因随着这些目标的完成，我们将拥有 系统地评估了三种用于抑制有害重复扩增的互补方法 C9orf72 基因：(1) 仅包含重复扩增的非编码 DNA 的双等位基因切除（目标 1），(2) 等位基因 特异性切除含有重复扩增的突变等位基因（目标 1），(3) 调节区破坏 选择性地沉默 C9orf72 重复扩展（目标 3）。 纠正与疾病相关的细胞类型（人类皮质和运动神经元）的疾病病理学的策略。 我们开发了快速而强大的方法来从人类诱导多能干细胞中产生神经元 对编辑的对照细胞系进行分析将使我们能够筛选来自对照和患者的（iPSC）。 精确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研究结果不仅会产生意想不到的影响。 促进我们对潜在治疗方法的理解，同时也将有助于我们理解 C9orf72 生物学，包括 C9orf72 基因调控和疾病的潜在机制。 将开发一个管道来系统地评估具有潜在疗效的编辑策略。