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

项目总结/摘要 信号转导子和转录激活子3（STAT 3）功能获得（GOF）种系突变是一种遗传性疾病。 早发性多器官自身免疫性和淋巴增生性疾病综合征的单基因原因， 目前唯一确定的治愈方法是异基因造血干细胞移植。尽管显然 对该综合征的遗传学理解，对该综合征的细胞和分子机制理解 仍然是模糊的，特别是关于STAT 3 GOF对T细胞的影响，这种细胞类型被认为是 导致STAT 3 GOF患者中出现的大部分自身免疫。该提案是一项计划的一部分， 项目补助金，旨在获得机械深入了解这种疾病的免疫病因， 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STAT 3 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革命。科学家现在拥有 测试人类基因序列的细胞功能和纠正突变的前所未有的能力， 直接在小鼠和人类的原代细胞中引起疾病。与本项目提案相关，我们的实验室 开创性的方法和系统，以在来自人的原代T细胞中进行靶向和全基因组敲除筛选， 老鼠和人类此外，我们已经开发了一种使用Cas9进行正交验证的有效方法： 向导RNA核糖核蛋白（Cas9 RNP）电穿孔与多重流式细胞术表型分析偶联。 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STAT 3 GOF综合征中异常T细胞功能的新途径，优先考虑进一步研究的新靶点， 研究药物开发，并改善基因重编程细胞为基础的疗法的设计。 具体来说，项目3旨在在小鼠T细胞中实施无偏见的CRISPR功能遗传方法 模拟STAT 3 GOF突变以鉴定调节STAT 3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的信息来改善人类的功能。 患者T细胞（Aim 1c）。最后，项目3寻求使用基于非病毒CRISPR的尖端方法来纠正 STAT 3 GOF突变在实际的患者T细胞，以及开发一种临床前方法，以取代选择的 原代人造血干祖细胞（HSPC）中的STAT 3外显子（Aim 2）。综上所述各项 这些研究将为理解STAT 3 GOF变体如何改变T细胞功能以驱动 炎症性疾病以及打开多种途径免疫细胞CRISPR基因靶向治疗 自身免疫

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.