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.

抽象的嵌合抗原受体（CAR）T细胞疗法已被证明是一种突破性的治疗方法，具有治愈性然而，最近的研究表明，它在血液癌症患者以及侵袭性临床前模型中具有潜力。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会复发，CAR T细胞在治疗实体瘤方面已证明临床疗效有限导致这些失败模式的一个关键现象是 CAR T 细胞耗竭，强效信号 CAR T 细胞被驱动为一种独特且功能失调的表型，且受到限制之前使用 CRISPR-Cas9 进行的全基因组扰动研究已经确定了越来越多的抗肿瘤活性。单基因靶点被敲除后，有助于减轻疲劳并适度改善 T 细胞功能。然而，这些筛选产生的个体基因命中通常是依赖于环境的，并且在不同的环境中是不同的。总而言之，这些研究表明，耗尽的 T 细胞表型很大程度上是由不同的肿瘤和 CAR 模型决定的。由关键基因程序而不是单个基因的上调驱动，尽管这些程序的复杂网络遗传相互作用仍然不明确。为了解决这些未满足的需求，我们提出了一种使用 CRISPR-Cas13d 的合成生物学驱动方法 Cas13d 是一种小型 CRISPR RNA- 转录组工程，用于开发有效的、抗耗竭的 CAR T 细胞。引导RNA核酸内切酶，可以处理单个引导RNA阵列以降解多个不同的靶RNA 在 AIM 1 中，我们将使用高度序列特异性和稳健的方式开发一个新颖的平台。 Cas13d 可同时下调人类原代 T 细胞中的多个内源基因，具有特定的专注于改善耗尽的CAR T细胞效应功能。在AIM 2中，我们将利用该技术进行一项研究。针对假定负调节因子对的耗竭 CAR T 细胞的双重敲低增殖筛选我们将利用已建立的计算框架来识别高度富集的 T 细胞抗肿瘤活性。基因配对，绘制基因之间的遗传相互作用（GI），并定义耗尽网络。我们的多模式筛选方法可用于识别新的协同基因配对优于先前研究中发现的单一敲低表型。我们提出的项目将建立一个新的平台，用于初级阶段的多重基因抑制和筛查人类 T 细胞克服了最先进的 CRISPR-Cas9 和 RNAi 技术所面临的限制。此外，我们的研究将展示一种减轻 CAR T 细胞耗竭的新策略，这将改善最终，我们的工作将解决临床未满足的问题。需求并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。