CRISPR-Cas9 editing in C9orf72 patient derived iPSC

父项目摘要/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)的常见遗传原因，两人死亡 和不可逆转的神经退行性疾病，没有有效的治疗。考虑到细胞功能障碍 由这种突变引起的是多因素的，通过CRISPR/Cas9基因编辑来靶向基因本身是一个潜在的 治疗性干预。 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患者的C9ORF72病基因来源于IPSC。将评估三种编辑策略的能力 纠正病理性C9orf72重复扩增，包括：a)切除外显子1a以沉默转录 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 (目标1a)。此外，基因编辑的效率、偏离目标的效果以及纠正细胞病理的能力 将在C9FTD/ALS患者来源的神经元和小胶质细胞中进行研究(目标1b)。最后，新单曲- 将采用分子测序技术准确测量重复扩展大小，以确定 在不同的患者IPSC系和人体组织中重复扩增(目标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 生物信息学来解决这些目标。 我寻求K08临床科学家导师研究职业发展奖的目标是获得 作为一名R01资助的独立学者，开始我的职业生涯所需的知识和科学培训 调查员。我的长期目标是在治疗神经退行性变的方法上取得重大进展。 疾病，包括痴呆症。我的博士和临床培训为我提供了解决问题的动力 临床相关和高影响力假说驱动的研究问题。特殊的研究 加州大学旧金山分校附属格莱斯顿学院的环境将许多领域的领导者与尖端技术结合在一起 技术和设备。我已经组建了一支由非常有成就的导师和顾问组成的团队来指导 我走上了成为独立调查员的道路。我的训练计划是为了提高我的技术水平 对我来说是新领域的技能(CRISPR基因编辑、基因组分析、生物信息学和高级 细胞疾病建模)，以及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集 这将使我做好准备，获得R01或同等的资金，开始我的独立职业生涯 调查员。

PARENT 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.