CRISPR-Cas9 editing in C9orf72 patient derived iPSC

C9orf72 患者来源的 iPSC 中的 CRISPR-Cas9 编辑

基本信息

批准号：
10192850
负责人：
Claire Clelland
金额：
$ 21.33万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10192850
关键词：
ALS patients Address Alleles Area Autopsy Award Bioinformatics Biological Sciences Brain C9ORF72 CRISPR therapeutics CRISPR/Cas technology Cell Line Cell Survival Cell model Cell physiology Cells Clinical Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Communication DNA DNA sequencing Dementia Disease Disease model Elements Engineering Environment Equipment Exons Functional disorder Funding Gene Expression Gene Expression Regulation Gene Silencing Genes Genetic Genetic Transcription Genomics Goals Guide RNA Heritability Human Individual Institutes K-Series Research Career Programs Knowledge Link Measures Mendelian disorder Mentors Mentorship Methods Microglia Modeling Molecular Motor Neurons Mutation Neurodegenerative Disorders Neuroglia Neurons Pathogenicity Pathologic Pathology Patients Phase Regulation Research Research Personnel Research Training Role Running Scientist Specificity Spinal Cord Technical Expertise Technology Testing Therapeutic Tissues Training Transcription Initiation Site Work c9FTD/ALS career cell type cellular pathology clinically relevant curative treatments design effective therapy experience frontotemporal lobar dementia-amyotrophic lateral sclerosis genome analysis human disease human tissue induced pluripotent stem cell innovation mutant mutation carrier novel novel therapeutic intervention novel therapeutics promoter single molecule skills therapeutic gene therapeutic genome editing tool whole genome

ALS patients Address Alleles Area Autopsy Award Bioinformatics Biological Sciences Brain C9ORF72 CRISPR therapeutics CRISPR/Cas technology Cell Line Cell Survival Cell model Cell physiology Cells Clinical Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Communication DNA DNA sequencing Dementia Disease Disease model Elements Engineering Environment Equipment Exons Functional disorder Funding Gene Expression Gene Expression Regulation Gene Silencing Genes Genetic Genetic Transcription Genomics Goals Guide RNA Heritability Human Individual Institutes K-Series Research Career Programs Knowledge Link Measures Mendelian disorder Mentors Mentorship Methods Microglia Modeling Molecular Motor Neurons Mutation Neurodegenerative Disorders Neuroglia Neurons Pathogenicity Pathologic Pathology Patients Phase Regulation Research Research Personnel Research Training Role Running Scientist Specificity Spinal Cord Technical Expertise Technology Testing Therapeutic Tissues Training Transcription Initiation Site Work c9FTD/ALS career cell type cellular pathology clinically relevant curative treatments design effective therapy experience frontotemporal lobar dementia-amyotrophic lateral sclerosis genome analysis human disease human tissue induced pluripotent stem cell innovation mutant mutation carrier novel novel therapeutic intervention novel therapeutics promoter single molecule skills therapeutic gene therapeutic genome editing tool whole genome

展开

项目摘要

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.

项目摘要/摘要在C9ORF72基因的单个等位基因中，杂合六核苷酸（GGGGCC）重复膨胀是最多的额颞痴呆（FTD）和肌萎缩性侧面硬化症（ALS）的频繁遗传原因，两个致命和不可逆的神经退行性疾病，无需有效治疗。鉴于细胞功能障碍由此突变引起的是多因素，通过CRISPR/CAS9基因编辑针对基因本身是一种潜在的治愈性干预。这项工作提出了CRISPR基因编辑技术的新颖应用来编辑或使致病性静音 FTD/ALS患者衍生的IPSC中的C9ORF72疾病基因。将评估三种编辑策略的能力纠正病理C9orf72重复扩展，包括：a）切除外显子1a以沉默的转录重复膨胀，b）切除重复膨胀本身，c）等位基因特异性的突变等位基因灭活（AIM 1A）。另外，基因编辑的效率，关闭目标效应以及纠正细胞病理的能力将在C9FTD/ALS患者衍生的神经元和小胶质细胞中进行研究（AIM 1B）。最后，新的单人分子测序技术准确测量重复扩展大小将用于大小在各种患者的IPSC线和人体组织中重复扩展（AIM 2）。这项工作结合了小说 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以解决这些目标。我寻求K08指导的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，以启动我作为独立R01资助的学术的职业生涯研究者。我的长期目标是在神经退行性的治疗方法中取得重大进步疾病，包括痴呆症。我的博士和临床培训为我提供了解决的动力临床相关和高影响假设驱动的研究问题。杰出研究 UCSF附属的Gladstone Institutes的环境将许多领域的领导者与尖端结合在一起技术和设备。我组建了一个由高度成就的导师和顾问团队来指导我正在成为成为独立调查员的道路。我的培训计划旨在提高我的技术在我新的领域中设定的技能（CRISPR基因编辑，基因组分析，生物信息学和高级细胞疾病建模）以及在成功运行实验室至关重要的领域提供经验（科学沟通和实验室管理）。完成研究并获得我建议的技能这里会为我做好准备，以获得R01或同等资金，以启动我作为独立的职业研究者。