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：
10328103
负责人：
Alexander Marson
金额：
$ 47.25万
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/10328103
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 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 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 base cell type curative treatments cytokine design disease-causing mutation drug development early onset experimental study functional genomics gain of function gain of function mutation gene correction gene discovery gene interaction gene therapy genetic approach genome editing genome wide screen genome-wide immune function improved improved functioning in vivo inhibitor insight mouse model 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.

项目摘要/摘要转录3（STAT3）功能（GOF）种系突变的信号换能器和激活因子是早期发作多机器人自身免疫性和淋巴增生性疾病的综合征的单基因原因当前唯一的确定治疗是一种同种异体造血干细胞移植。尽管很清楚对该综合征，机械细胞和分子理解对该综合征的遗传理解仍然晦涩难懂，特别是关于STAT3 GOF对T细胞的影响，该细胞类型被认为是负责在STAT3 GOF患者中看到的许多自身免疫。该建议是计划的一部分旨在获得机械洞察的项目赠款，以了解这种疾病的免疫学病因人类患者样品，机械小鼠研究，T细胞培养实验和功能基因组学方法。计划赠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，以了解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遗传序列的细胞功能并纠正突变的空前功能直接在原代小鼠和人类细胞中引起疾病。与该项目建议相关，我们的实验室有开创性的方法和系统，以从老鼠和人类。此外，我们已经开发了一种有效的方法，用于使用Cas9：单一验证：引导RNA核糖核蛋白（Cas9 RNP）电穿孔与多重流式细胞仪表型结合。这些进步现在定位我们在小鼠和人类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T细胞功能异常T细胞功能的新型途径，将新目标优先考虑药物开发研究，并改善基于基于细胞的遗传学重编程的设计。具体而言，项目3旨在在小鼠T细胞中实施无偏的CRIS功能遗传方法对STAT3 GOF突变进行建模以识别调节STAT3介导的炎症的关键基因靶标（AIM 1A，1B）。此外，我们试图利用这些屏幕中的信息来改善人类的功能患者T细胞（AIM 1C）。最后，项目3旨在使用尖端的非病毒CRISPR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以替代选定的方法原发性人类造血干细胞（HSPC）中的STAT3外显子（AIM 2）。总的来说，这些研究将构成一个框架的基础，以了解STAT3 GOF变体如何改变T细胞函数以驱动炎症性疾病以及开放多种途径朝着治疗的免疫细胞CRISPR基因靶向自身免疫性。