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

摘要 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，可治愈 在血液病患者和侵袭性临床前模型中具有潜在的应用价值。然而，最近的研究 揭示了进展的主要障碍-许多患者最初对CAR T细胞完全应答 治疗最终复发，CAR T细胞在治疗实体瘤中的临床疗效有限 肿瘤。在这些故障模式中因果关联的一个关键现象是CAR T细胞耗尽， 其中，张力信号CAR T细胞被驱动到一种独特的、功能失调的表型，并受到抑制 抗肿瘤活性。之前使用CRISPR-Cas9进行的全基因组扰动研究已经确定了一个不断增长的清单 单基因靶点，当被敲除时，有助于缓解疲惫并适度改善T细胞功能。 然而，从这些屏幕上得到的单个基因点击通常是与上下文相关的，并且在 不同的肿瘤和汽车型号。总之，这些研究表明，耗尽的T细胞表型在很大程度上是 由关键基因程序的上调驱动，而不是单基因，尽管这些基因的复杂网络 基因的相互作用仍然没有得到很好的定义。 为了解决这些未得到满足的需求，我们提出了一种使用CRISPR-Cas13d的合成生物学驱动方法 转录组工程以开发强大的、抗疲劳的CAR T细胞。Cas13d是一个小的CRISPR RNA- 可处理单个引导RNA阵列以降解多个不同目标RNA的引导RNA内切酶 以高度序列特异性和健壮的方式转录。在目标1中，我们将开发一个新的平台，使用 Cas13d同时下调原代人类T细胞中的多个内源性基因，具有特异性 重点提高耗竭汽车T细胞效应器功能。在AIM 2中，我们将使用这项技术进行 针对可能的负性调节因子对耗尽的CAR T细胞进行双击倒增殖筛选 T细胞的抗肿瘤活性。我们将利用已建立的计算框架来识别高度浓缩的 基因配对，以绘制基因之间的遗传相互作用(GI)图，并定义耗竭网络。我们 假设我们的多模式筛选方法可以用于识别新的协同基因配对 这比之前研究中看到的单一基因敲除表型表现更好。 我们提出的项目将建立一个新的平台，用于多重基因抑制和初级筛查 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新的策略来缓解CAR T细胞耗竭，这将改善 对目前的免疫疗法进行研究，并提高其有效性。最终，我们的工作将解决临床上未得到满足的问题 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)使用这些数据来为下一代CAR T细胞疗法的开发提供信息。

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.