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

• 批准号: 10431227
• 负责人: Lei Stanley Qi
• 金额: $ 18.29万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10431227
• 关键词: Address; Biological; Biological Assay; CAR T cell therapy; CRISPR/Cas technology; Cancer Patient; Cell physiology; Chromosomal translocation; Clinical; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Computer Analysis; Custom; Data; Development; 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; Light; Maps; Methodology; Methods; Modeling; Outcome; Patients; Phenotype; Post-Transcriptional Regulation; Pre-Clinical Model; Process; 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; base; 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; screening; small molecule; synthetic biology; transcriptome; tumor

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治疗性的突破性治疗 血液学癌症患者以及侵略性临床前模型的潜力。但是，最近的研究 已经阐明了进步的主要障碍 - 许多最初对汽车T细胞完全反应的患者 治疗最终中继，CAR T细胞在固体治疗方面表现出有限的临床效率 肿瘤。在这些故障模式中随便涉及的一个关键现象是汽车T细胞耗尽， 固有信号的汽车T细胞被驱动到具有约束的独特且功能障碍的表型 抗肿瘤活性。以前使用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 单个基因靶向，当被淘汰时，有助于减轻疲劳并适度改善T细胞功能。 但是，从这些筛选中产生的个体基因命中通常依赖于上下文 不同的肿瘤和汽车模型。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 遗传相互作用的定义较差。 为了满足这些未满足的需求，我们建议使用CRISPR-CAS13D进行合成生物学驱动的方法 转录组工程以开发潜在的，耗尽的汽车T细胞。 CAS13D是一个小CRISPR RNA- 可以处理单个指南RNA阵列以降解多个不同靶RNA的引导RNA内切核酸酶 以高度序列特异性和鲁棒的方式进行转录本。在AIM 1中，我们将使用一个新颖的平台使用 CAS13D简单地下调原代人T细胞中的多个内源基因，具体 专注于改善耗尽的CAR T细胞效应器功能。在AIM 2中，我们将使用这项技术进行 耗尽的汽车T细胞中的双重敲低增殖屏幕瞄准了推定的负调节器 T细胞抗肿瘤活性。我们将利用既定的计算框架来识别高度丰富的 基因配对，以绘制基因之间的遗传相互作用（GI），并定义疲惫网络。我们 假设我们的多模式筛选方法可用于识别新的协同基因配对 在先前的研究中，这种表现优于单一敲低表型。 我们提出的项目将建立一个新的平台，用于多路复用基因表达和筛选。 由最先进的CRISPR-CAS9和RNAi技术所面临的限制的人类T细胞。 此外，我们的研究将证明一种减轻汽车T细胞耗尽的新型策略，这将改善 在当前的免疫疗法并增强其有效性。最终，我们的工作将无法解决临床 需求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 疲惫和2）使用此数据来告知下一代CAR T细胞疗法的发展。