CRISPR-Cas9 editing in C9orf72 patient derived iPSC

项目总结/摘要 在C9 orf 72基因的单个等位基因中杂合六核苷酸（GGGGCC）重复扩增是最常见的。 额颞叶痴呆症（FTD）和肌萎缩侧索硬化症（ALS）的常见遗传原因，两种致命的 和不可逆的神经退行性疾病而得不到有效治疗。考虑到细胞功能障碍 这种突变引起的基因突变是多因素的，通过CRISPR/Cas9基因编辑靶向基因本身是一种潜在的 治疗性干预 这项工作提出了CRISPR基因编辑技术的新应用，以编辑或沉默致病基因。 FTD/ALS患者来源的iPSC中的C9 orf 72疾病基因。将评估三种编辑策略的能力， 纠正病理性C9 orf 72重复扩增，包括：A）切除外显子1a以沉默C9 orf 72的转录， 重复扩增，B）切除重复扩增本身，C）突变等位基因的等位基因特异性失活 (Aim 1a）。此外，基因编辑的效率、脱靶效应以及纠正细胞病理的能力 将在C9 FTD/ALS患者源性神经元和小胶质细胞中进行研究（目的1b）。最后，新单曲- 将采用精确测量重复扩增大小的分子测序技术来确定重复扩增的大小。 在各种患者iPSC系和人组织中重复扩增（目的2）。这部作品结合了小说 CRISPR/Cas9编辑技术、细胞疾病建模、分子和基因组分析，以及 生物信息学来实现这些目标。 我寻求K 08指导临床科学家研究职业发展奖的目标是获得 必要的知识和科学培训，以启动我的职业生涯作为一个独立的R 01资助的学者 调查员我的长期目标是在神经退行性疾病的治疗方法上取得重大进展， 疾病，包括痴呆症。我的博士和临床训练为我提供了解决问题的动力， 临床相关和高影响假设驱动的研究问题。卓越的研究 在加州大学旧金山分校附属格莱斯顿研究所的环境结合了许多领域的领导者与尖端 技术和设备。我召集了一个由高水平的导师和顾问组成的团队 我正在成为一名独立调查员我的培训计划旨在提高我的技术水平 我在新领域的技能（CRISPR基因编辑，基因组分析，生物信息学和高级 细胞疾病建模），并提供成功运行实验室的关键领域的经验 （科学交流和实验室管理）。完成研究并获得我建议的技能 这里将准备我很好地获得R 01或同等的资金，以启动我的职业生涯作为一个独立的 调查员

PROJECT SUMMARY/ABSTRACT A heterozygous hexanucleotide (GGGGCC) repeat expansion in a single allele of the C9orf72 gene is the most frequent genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal and irreversible neurodegenerative diseases without effective treatment. Given that the cellular dysfunction caused by this mutation is multifactorial, targeting the gene itself by CRISPR/Cas9 gene editing is a potentially curative intervention. This work proposes novel applications of CRISPR gene editing technology to edit or silence the pathogenic C9orf72 disease gene in FTD/ALS patient derived iPSC. Three editing strategies will be evaluated for ability to correct the pathologic C9orf72 repeat expansion, including: A) excising exon 1a to silence transcription of the repeat expansion, B) excising the repeat expansion itself, C) allele-specific inactivation of the mutant allele (Aim 1a). In addition, the efficiency of gene editing, off target effects, and ability to correct cellular pathology will be investigated in C9FTD/ALS patient-derived neurons and microglia (Aim 1b). Finally, new single- molecule sequencing technology to accurately measure the repeat expansion size will be employed to size the repeat expansion in various patient iPSC lines and human tissue (Aim 2). This work combines novel CRISPR/Cas9 editing technologies, cellular disease modeling, molecular and genomic analysis, and bioinformatics to address these aims. My goal in seeking a K08 Mentored Clinical Scientist Research Career Development Award is to acquire the necessary knowledge and scientific training to launch my career as an independent R01 funded academic investigator. My long-term goal is to make major advances in the therapeutic approaches to neurodegenerative diseases, including dementias. My doctoral and clinical training provide me with the impetus to address clinically relevant and high impact hypothesis driven research questions. The exceptional research environment at the UCSF affiliated Gladstone Institutes combines leaders across many fields with cutting-edge technology and equipment. I have assembled a team of highly accomplished mentors and advisors to guide me on my path to becoming an independent investigator. My training plan is designed to advance my technical skill set in areas that are new to me (CRISPR gene editing, genomic analysis, bioinformatics, and advanced cellular disease modeling) as well as provide experience in areas critical to successfully running a lab (scientific communication and lab management). Completing the research and obtaining the skill set I propose here will prepare me well to obtain an R01 or equivalent funding to launch my career as an independent investigator.