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Project 3: CRISPR Genome Editing to Understand and Correct STAT3 GOF Immune Dysregulation

项目 3：通过 CRISPR 基因组编辑了解和纠正 STAT3 GOF 免疫失调

基本信息

批准号：
10576392
负责人：
Alexander Marson
金额：
$ 47.16万
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：
2022
资助国家：
美国
起止时间：
2022-02-17 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10576392
关键词：
Ablation Affect Allogenic Animals Autoimmune Diseases Autoimmunity Automobile Driving Biology CRISPR screen CRISPR/Cas technology Cell Communication Cell Culture System Cell Culture Techniques Cell Differentiation process Cell Survival Cell Therapy Cell model Cell physiology Cells Chemosensitization Clustered Regularly Interspaced Short Palindromic Repeats Complex Coupled Critical Pathways DNA Sequence Development Disease Electroporation Epistatic Gene Etiology Exons Family Future Gene Targeting Genes Genetic Genetic Screening Genetic Transcription Genome Genome engineering Genomic approach Genomics Germ-Line Mutation Grant Guide RNA Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Heterozygote Human Human Genetics IL6 gene Imiquimod Immune Immune Tolerance Immune response Immune system Immunological Models Immunologics In Vitro Inflammation Inflammatory Investigation Knock-out Libraries Lymphoproliferative Disorders Maps Mediating Methods Modeling Modernization Molecular Mus Mutation Organ Pathogenesis Pathogenicity Pathology Pathway interactions Patients Peripheral Phenotype Physiological Positioning Attribute Program Research Project Grants Regulatory T-Lymphocyte Ribonucleoproteins Sampling Scientist Signal Transduction Site Skin Source Stat3 protein Surveys Syndrome System T cell response T-Lymphocyte Technology Testing Therapy Clinical Trials Time Validation Variant Work cell type curative treatments cytokine design disease-causing mutation drug development early onset experimental study functional genomics functional improvement gain of function gene correction gene discovery gene interaction gene therapy genetic approach genome editing genome wide screen genome-wide improved in vivo inhibitor insight mouse model mutation correction novel novel therapeutics pre-clinical preservation programs synergism therapeutic target whole genome

项目摘要

Project Summary/Abstract Signal transducer and activator of transcription 3 (STAT3) gain of function (GOF) germline mutations are a monogenic cause of a syndrome of early-onset multiorgan autoimmune and lymphoproliferative disease for which the only current definitive cure is an allogeneic hematopoietic stem cell transplant. Despite the clear genetic understanding of this syndrome, mechanistic cellular and molecular understanding of this syndrome remains obscure, particularly with regards to the STAT3 GOF effects on T cells, the cell type thought to be responsible for much of the autoimmunity seen in STAT3 GOF patients. This proposal is part of a Program Project Grant that seeks to gain mechanistic insight into the immunological etiology of this disease incorporating human patient samples, mechanistic mouse studies, T cell culture experiments, and functional genomics approaches. This specific Project within the Program Grant seeks to leverage functional genomics approaches powered by CRISPR-Cas9 technology to understand how STAT3 GOF variants affect T cell function. CRISPR- Cas9 genome engineering technology is driving a revolution in modern biology. Scientists now possess unprecedented capabilities to test the cellular functions of human genetic sequences and correct mutations that cause disease directly in primary mouse and human cells. Of relevance to this Project proposal, our lab has pioneered methods and systems to conduct targeted and genome-wide knockout screens in primary T cells from mice and humans. Further, we have developed an efficient method for orthogonal validations using Cas9:single- guide RNA ribonucleoprotein (Cas9 RNP) electroporation coupled with multiplexed flow cytometric phenotyping. These advances now position us to use functional genomics approaches in mouse and human T cells to reveal novel pathways that control abnormal T cell function in STAT3 GOF syndrome, prioritize novel targets in further studies for drug development, and improve the design of genetically reprogrammed cell-based therapies. Specifically, Project 3 seeks to implement unbiased CRISPR functional genetic approaches in mouse T cells modeling STAT3 GOF mutations to identify critical gene targets that modulate STAT3-mediated inflammation (Aim 1a, 1b). Further, we seek to leverage the information from these screens to improve function in human patient T cells (Aim 1c). Finally, Project 3 seeks to use cutting-edge non-viral CRISPR based methods to correct STAT3 GOF mutations in actual patient T cells, as well as to develop a preclinical approach to replace selected STAT3 exons in primary human hematopoietic stem progenitor cells (HSPCs) (Aim 2). Taken together, these studies will form the basis for a framework to understand how STAT3 GOF variants alter T cell function to drive inflammatory disease as well as open multiple avenues towards immune cell CRISPR gene targeting to treat autoimmunity.

项目概要/摘要信号转导子和转录激活子 3 (STAT3) 功能获得性 (GOF) 种系突变是早发性多器官自身免疫性和淋巴组织增生性疾病综合征的单基因原因目前唯一确定的治疗方法是同种异体造血干细胞移植。尽管明确对这种综合征的遗传学理解，对这种综合征的机制细胞和分子理解仍然不清楚，特别是关于 STAT3 GOF 对 T 细胞的影响，该细胞类型被认为是 STAT3 GOF 患者中出现的大部分自身免疫性疾病都是由其引起的。该提案是计划的一部分项目资助旨在深入了解这种疾病的免疫学病因，包括人类患者样本、机制小鼠研究、T 细胞培养实验和功能基因组学接近。该计划拨款中的这个特定项目旨在利用功能基因组学方法由 CRISPR-Cas9 技术提供支持，了解 STAT3 GOF 变体如何影响 T 细胞功能。 CRISPR- Cas9基因组工程技术正在推动现代生物学的一场革命。科学家现在拥有测试人类基因序列的细胞功能并纠正突变的前所未有的能力直接在原代小鼠和人类细胞中引起疾病。与该项目提案相关，我们的实验室有开创了在原代 T 细胞中进行靶向和全基因组敲除筛选的方法和系统老鼠和人类。此外，我们还开发了一种使用 Cas9 进行正交验证的有效方法：single- 引导 RNA 核糖核蛋白 (Cas9 RNP) 电穿孔与多重流式细胞术表型分析相结合。这些进展现在使我们能够在小鼠和人类 T 细胞中使用功能基因组学方法来揭示控制 STAT3 GOF 综合征中异常 T 细胞功能的新途径，进一步优先考虑新靶标研究药物开发，并改进基于基因重编程的细胞疗法的设计。具体来说，项目 3 寻求在小鼠 T 细胞中实施公正的 CRISPR 功能遗传方法模拟 STAT3 GOF 突变以确定调节 STAT3 介导的炎症的关键基因靶标（目标 1a、1b）。此外，我们寻求利用这些屏幕中的信息来改善人类的功能患者 T 细胞（目标 1c）。最后，项目 3 寻求使用基于尖端非病毒 CRISPR 的方法来纠正实际患者 T 细胞中的 STAT3 GOF 突变，以及开发临床前方法来替代选定的原代人造血干祖细胞 (HSPC) 中的 STAT3 外显子（目标 2）。综合起来，这些研究将为理解 STAT3 GOF 变体如何改变 T 细胞功能以驱动的框架奠定基础炎症性疾病以及为免疫细胞 CRISPR 基因靶向治疗开辟多种途径自身免疫。