权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

A Cas13d-based screening approach to engineer exhaustion-resistant CAR T cells

基于 Cas13d 的筛选方法来设计抗耗竭 CAR T 细胞

基本信息

批准号：
10571868
负责人：
Lei Stanley Qi
金额：
$ 21.61万
依托单位：
STANFORD UNIVERSITY
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：
2022-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10571868
关键词：
Address Biological Biological Assay CAR T cell therapy CRISPR/Cas technology Cancer Patient Cell physiology Chromosomal translocation Chromosome Mapping Clinical Clinical Research Clustered Regularly Interspaced Short Palindromic Repeats Communities Complex Computer Analysis Custom Data Development Disparate Effectiveness Endoribonucleases Engineering Epigenetic Process Failure Future Gene Library Gene Targeting Genes Genetic Genetic Transcription Guide RNA Hematologic Neoplasms Human Immunotherapy Impairment In Vitro Individual Knock-out Maps Methodology Methods Modeling Outcome Patients Phenotype Post-Transcriptional Regulation Pre-Clinical Model Process Proliferating RNA RNA Interference Relapse Repression Resistance Risk Signal Transduction Solid Neoplasm Specificity System T cell therapy T-Lymphocyte Technology Transcript Up-Regulation Validation Variant Work cancer type chimeric antigen receptor chimeric antigen receptor T cells clinical efficacy computer framework cytokine cytotoxicity design effector T cell engineered T cells exhaust exhaustion gene repression genetic analysis genome-wide genotoxicity improved knock-down multimodality next generation novel novel strategies programmed cell death protein 1 programs restraint screening small molecule synthetic biology transcriptome tumor

项目摘要

ABSTRACT Chimeric Antigen Receptor (CAR) T cell therapy has proven to be a breakthrough treatment with curative potential in hematologic cancer patients as well as in aggressive preclinical models. However, recent studies have shed light on major barriers to progress – many patients that initially respond completely to CAR T cell therapy eventually relapse, and CAR T cells have demonstrated limited clinical efficacy in the treatment of solid tumors. A key phenomenon that has been causally implicated in these failure modes is CAR T cell exhaustion, where tonically-signaling CAR T cells are driven to a distinct and dysfunctional phenotype with restrained antitumor activity. Previous genome-wide perturbation studies using CRISPR-Cas9 have identified a growing list of single gene targets that, when knocked out, help mitigate exhaustion and modestly improve T cell function. However, the resulting individual gene hits from these screens are often context-dependent and disparate across different tumor and CAR models. Altogether, these studies indicate that the exhausted T cell phenotype is largely driven by the upregulation of key gene programs rather than single genes, though the complex network of these genetic interactions remains poorly defined. To address these unmet needs, we propose a synthetic biology-driven approach using CRISPR-Cas13d transcriptome engineering to develop potent, exhaustion-resistant CAR T cells. Cas13d is a small CRISPR RNA- guided RNA endonuclease that can process a single guide RNA array to degrade multiple distinct target RNA transcripts in a highly sequence-specific and robust manner. In AIM 1, we will develop a novel platform using Cas13d to simultaneously downregulate multiple endogenous genes in primary human T cells, with a specific focus on improving exhausted CAR T cell effector function. In AIM 2, we will use this technology to conduct a double knockdown proliferation screen in exhausted CAR T cells targeting pairs of putative negative regulators of T cell antitumor activity. We will utilize an established computational framework to identify highly enriched gene pairings, to map genetic interactions (GI) between genes, and to define a network of exhaustion. We hypothesize that our multimodal screening methodology can be used to identify new synergistic gene pairings that outperform single knockdown phenotypes seen in prior studies. Our proposed project will establish a new platform for multiplexed gene repression and screening in primary human T cells that overcomes limitations faced by state-of-the-art CRISPR-Cas9 and RNAi technologies. Furthermore, our studies will demonstrate a novel strategy to mitigate CAR T cell exhaustion, which will improve upon current immune therapies and enhance their effectiveness. Ultimately, our work will address clinical unmet needs as well as help the broader scientific community 1) better understand the complex network of T cell exhaustion and 2) use this data to inform the development of next-generation CAR T cell therapies.

摘要嵌合抗原受体（CAR）T细胞疗法已被证明是具有治愈性的突破性治疗。在血液癌症患者以及侵袭性临床前模型中的潜力。但最近的研究已经揭示了进展的主要障碍-许多患者最初对CAR T细胞完全反应，治疗最终复发，并且CAR T细胞在治疗实体瘤中表现出有限的临床疗效。肿瘤的与这些失效模式有因果关系的一个关键现象是CAR T细胞耗竭，其中紧张性信号传导CAR T细胞被驱动为具有受限制的功能障碍的独特表型，抗肿瘤活性。之前使用CRISPR-Cas9的全基因组扰动研究已经确定了一个不断增长的列表单基因靶点，当敲除时，有助于减轻疲惫并适度改善T细胞功能。然而，从这些筛选中得到的单个基因命中通常是依赖于上下文的，并且在不同的基因组中是不同的。不同的肿瘤和CAR模型。总之，这些研究表明，耗尽的T细胞表型在很大程度上是由关键基因程序而不是单个基因的上调驱动，尽管这些基因的复杂网络基因间的相互作用仍不明确。为了解决这些未满足的需求，我们提出了一种使用CRISPR-Cas 13 d的合成生物学驱动的方法。转录组工程以开发有效的耐耗竭CAR T细胞。Cas 13 d是一种小CRISPR RNA。引导RNA核酸内切酶，其可以处理单个引导RNA阵列以降解多个不同的靶RNA 以高度序列特异性和稳健的方式转录。在AIM 1中，我们将使用 Cas 13 d同时下调原代人T细胞中的多种内源性基因，具有特异性的专注于改善耗尽的CAR T细胞效应子功能。在AIM 2中，我们将使用该技术进行在靶向假定的负调节物对的耗尽的CAR T细胞中进行双敲低增殖筛选 T细胞的抗肿瘤活性。我们将利用一个已建立的计算框架来识别高度富集的基因配对，绘制基因之间的遗传相互作用（GI），并定义一个耗尽网络。我们假设我们的多模式筛选方法可用于鉴定新的协同基因配对其表现优于先前研究中所见的单一敲除表型。我们的项目将建立一个新的平台，在初级阶段进行多重基因抑制和筛选。人类T细胞，克服了最先进的CRISPR-Cas9和RNAi技术所面临的限制。此外，我们的研究将证明一种减轻CAR T细胞耗竭的新策略，并增强其有效性。最终，我们的工作将解决临床未满足的需要以及帮助更广泛的科学界1）更好地了解T细胞的复杂网络 2）使用这些数据为下一代CAR T细胞疗法的开发提供信息。