Endogenous T Cell Receptor Replacement in Autoimmune Diabetes

项目总结/摘要 人类T细胞是生理免疫稳态的核心，它保护我们免受病原体的侵害， 自身免疫性炎症T细胞的多克隆群体已被用作癌症疗法 （肿瘤浸润淋巴细胞），以及最近的1型糖尿病（T1 D）中的多克隆调节性T细胞（TlD）。 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）或嵌合抗原受体来工程化抗原特异性 在人类癌症试验和小鼠肿瘤模型中， 1型糖尿病这种工程化可以通过使用逆转录病毒载体来完成，并且最近基因组 编辑带来了特异性和有效插入大转基因的希望。然而这些 这些方法仍然需要病毒转导，减缓了研究和临床应用。为了克服这些局限性， 我的初步工作是开发了一种新型的非病毒CRISPR-Cas9基因组靶向系统， 在特定位点快速有效地插入单个或多个大（>1个内切酶）DNA序列 在原代人T细胞的基因组中，同时保留细胞活力和功能。我的第一个目标是， 使用这种非病毒基因组靶向系统来取代内源性TCR。这种方法将重新定向 治疗性T细胞的抗原特异性，同时维持其内源性TCR表达并使TCR最小化 错配对于我的第二个目标，我将展示用一种免疫调节剂替代调节性T细胞的内源性TCR， 使用非病毒基因靶向的T1 D自身抗原特异性TCR将产生更有效的和临床上更有效的TCR。 T1 D的可行细胞疗法我将演示重定向鼠标和人的能力 调节性T细胞识别确定的T1 D自身抗原并测定它们在体外和体内的功能性， 预防和逆转T1 D的发展。我的担保人亚历克斯·马森博士在 T1 D的遗传基础和原代T细胞的遗传工程;我的共同赞助人Mark安德森博士 对T1 D免疫耐受的本质有了基础性发现。除了我的两个赞助商， 我正在与Jeff Bluestone博士（领导多克隆调节T细胞的第一次临床试验）进行当地合作， 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，我也在接受纵向临床培训 与UCSF调节性T细胞治疗的医学主任Jonathan Esensten博士一起进行细胞治疗 组总之，本工作将为非病毒TCR替代在临床上的应用奠定基础。 调节性T细胞作为T1 D的治愈性细胞疗法。

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.