Endogenous T Cell Receptor Replacement in Autoimmune Diabetes

自身免疫性糖尿病中的内源性 T 细胞受体替代

基本信息

批准号：
9918151
负责人：
Theodore Lee Roth
金额：
$ 3.89万
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：
2018
资助国家：
美国
起止时间：
2018-09-14 至 2020-07-13
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9918151
关键词：
Adoptive Transfer Anti-Inflammatory Agents Antigenic Specificity Antigens Autoantigens Autoimmune Diabetes Autoimmune Diseases Autoimmune Process Automobile Driving Biological Assay CRISPR/Cas technology Cell Survival Cell Therapy Cell physiology Cells Cellular immunotherapy Clinical Clinical Trials Collaborations Complex DNA DNA Sequence Development Diabetes Mellitus Engineering Environment Exons Foundations Gene Targeting Genetic Genetic Engineering Genome Goals Homeostasis Human Immune Immune Tolerance In Vitro Inbred NOD Mice Individual Inflammation Insulin-Dependent Diabetes Mellitus Knock-in Knock-out Malignant Neoplasms Methods Modeling Mus Nature Patients Physician Executives Physiological Population Production Regulatory T-Lymphocyte Reproducibility Research Retroviral Vector Site Specificity System T cell therapy T-Cell Immunologic Specificity T-Cell Receptor T-Lymphocyte Testing Therapeutic Tissues Training Transgenes Tumor Antigens Tumor-Infiltrating Lymphocytes Viral Work cancer immunotherapy cancer therapy chimeric antigen receptor clinical application cytokine efficacy testing engineered T cells experimental study genome editing in vitro Assay in vivo islet mouse model novel pathogen preservation prevent promoter receptor receptor expression recombinant viral vector repaired synthetic biology therapeutic target tumor tumor xenograft

项目摘要

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.

项目概要/摘要人类 T 细胞是生理免疫稳态的核心，它可以保护我们免受病原体侵害，而无需附带的自身免疫性炎症。 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 1 型糖尿病 (T1D) 中的多克隆调节性 T 细胞 (Treg)。然而，绝大多数多克隆 T 细胞不能识别所需的肿瘤特异性抗原或自身抗原。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人类癌症试验和小鼠模型表明，（CAR）使转移的细胞更加有效 1 型糖尿病。这种工程可以通过使用逆转录病毒载体和最近的基因组来完成编辑带来了大规模转基因的特异性和高效插入的希望。然而这些方法仍然需要病毒转导，从而减缓了研究和临床使用。为了克服这些限制，在在我的前期工作中，我开发了一种新型非病毒 CRISPR-Cas9 基因组靶向系统，该系统允许在特定位点快速有效地插入单个或多重大（> 1 kilobase）DNA序列存在于原代人类 T 细胞的基因组中，同时保留细胞活力和功能。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替代内源性TCR。这种方法将重定向治疗性 T 细胞的抗原特异性，同时维持其内源性 TCR 表达并最大限度地减少 TCR 错配。对于我的第二个目标，我将展示用 TCR 替代调节性 T 细胞的内源性 TCR。使用非病毒基因靶向的 T1D 自身抗原特异性 TCR 将创造出更有效的临床药物 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 细胞识别特定的 T1D 自身抗原并测定其体外和体内功能预防和逆转 T1D 的发展。我的赞助商 Alex Marson 博士在以下方面拥有丰富的专业知识 T1D 的遗传基础和原代 T 细胞的基因工程；我的共同发起人马克·安德森博士对 T1D 免疫耐受的本质做出了基础性发现。除了我的两位赞助商之外，我与 Jeff Bluestone 博士（领导多克隆监管 T 的首次临床试验）正在进行的本地合作人类 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 Jonathan Esensten 博士一起研究细胞疗法团体。总体而言，这项工作将为非病毒TCR替代的临床应用奠定基础。调节性 T 细胞作为 T1D 的细胞疗法。