Endogenous T Cell Receptor Replacement in Autoimmune Diabetes

项目摘要/摘要 人类T细胞是生理免疫动态平衡的中心，它保护我们免受病原体的侵袭 伴随的自身免疫性炎症。T细胞的多克隆群体已被用于癌症治疗 (肿瘤浸润性淋巴细胞)，以及最近在1型糖尿病(T1D)中的多克隆调节性T细胞(Tregs)。 然而，绝大多数多克隆T细胞不识别所需的肿瘤特异性或自身抗原。 通过引入新的T细胞受体(TCR)或嵌合抗原受体来设计抗原特异性 (CARS)使转移的细胞更有效，在人类癌症试验和小鼠癌症模型中显示 1型糖尿病。这样的工程可以通过使用逆转录病毒载体来完成，最近还可以使用基因组 编辑带来了特定和有效插入大型转基因的前景。然而，这些 这些方法仍然需要病毒转导，减缓研究和临床应用。要克服这些限制，请在 我的初步工作我已经开发了一种新的非病毒CRISPR-Cas9基因组靶向系统，它允许 在特定位置快速有效地插入单个或多个大(1千碱基)DNA序列 在保持细胞活力和功能的同时，在原代人类T细胞的基因组中。在我的第一个目标中，我建议 使用这种非病毒基因组靶向系统来取代内源性TCR。此方法将重定向一个 治疗性T细胞在维持其内源性TCR表达和最小化TCR的同时的抗原特异性 配对错误。对于我的第二个目标，我将展示用一种 使用非病毒基因靶向的T1D自身抗原特异性TCR将创造更有效和临床 治疗T1D的可行细胞疗法。我将演示重定向鼠标和人类的能力 调节性T细胞识别确定的T1D自身抗原并检测其在体外和体内的功能 防止和逆转T1D的发展。我的赞助人亚历克斯·马森博士在 T1D的遗传基础和初级T细胞的基因工程；我的联合发起人马克·安德森博士 对T1D免疫耐受的本质做出了基础性的发现。除了我的两个赞助商， 我与Jeff Blustone博士正在进行的本地合作(领导多克隆调节性T细胞的第一次临床试验 人类T1D患者的细胞)和Kole Roybal博士(应用合成生物学重新设计T细胞特异性) 将进一步支持拟议工作的可行性。同样，我正在接受纵向临床培训 与加州大学旧金山分校调节性T细胞疗法医学主任乔纳森·埃森斯滕博士在细胞疗法中 组。总之，本工作将为非病毒TCR置换在临床上的应用奠定基础。 调节性T细胞作为一种治疗T1D的细胞疗法。

PROJECT SUMMARY/ABSTRACT Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. Polyclonal populations of T cells have been used as cancer therapies (tumor-infiltrating lymphocytes), and recently polyclonal regulatory T cells (Tregs) in type 1 diabets (T1D). However the vast majority of polyclonal T cells do not recognize a desired tumor specific or auto-antigen. Engineering antigen specificity by introduction of a new T cell receptor (TCR) or chimeric antigen receptor (CARs) makes the transferred cells much more potent, shown in human cancer trials and in mouse models of type 1 diabetes. Such engineering can be accomplished by using retroviral vectors, and recently genome editing has brought the promise of specific and efficient insertion of large transgenes. However these approaches still require viral transduction, slowing research and clinical use. To overcome these limitations, in my preliminary work I have developed a novel non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. In my first aim, I propose to use this non-viral genome targeting system to replace the endogenous TCR. This approach will redirect a therapeutic T cell's antigenic specificity while maintaining its endogenous TCR expression and minimizing TCR mispairing. For my second aim, I will show that replacement of regulatory T cell's endogenous TCR with a T1D autoantigen specific TCR using non-viral gene targeting will create a more potent and clinically viable cellular therapeutic for T1D. I will demonstrate the ability to redirect both mouse and human regulatory T cells to recognize a defined T1D autoantigen and assay their in vitro and in vivo functionality in preventing and reversing T1D development. My sponsor Dr. Alex Marson has extensive expertise in the genetic basis of T1D and the genetic engineering of primary T cells; my co-sponsor Dr. Mark Anderson has made foundational discoveries about the nature of immune tolerance in T1D. In addition to my two sponsors, my ongoing local collaborations with Dr. Jeff Bluestone (leading the first clinical trials of polyclonal regulatory T cells in human T1D patients) and Dr. Kole Roybal (applying synthetic biology to re-engineer T cell specificity) will further support the feasibility of the proposed work. Similarly, I am undergoing longitudinal clinical training in cellular therapeutics with Dr. Jonathan Esensten, the medical director of UCSF's Regulatory T Cell Therapy Group. Overall, this work will lay the foundation for clinical application of non-viral TCR replacement in regulatory T cells as a curative cellular therapy for T1D.