美国斯坦福大学Lei S. Qi和美国斯坦福大学医学院Crystal L. Mackall共同合作，近期取得重要工作进展。他们研究开发了用于原代人类T细胞多重转录组调控和代谢工程的多功能CRISPR-Cas13d平台。相关研究成果2024年2月21日在线发表于《细胞》杂志上。
据介绍，CRISPR技术已经开始彻底改变T细胞疗法。然而，传统的CRISPR-Cas9基因组编辑工具在安全性、有效性和适用范围方面受到限制。
为了应对这些挑战，研究人员开发了多重效应器引导阵列（MEGA），这是一个使用CRISPR-Cas13d的RNA引导和RNA靶向活性对T细胞转录组进行可编程和可扩展调节的平台。MEGA能够在不靶向或切割基因组DNA的情况下，在原代人类T细胞中实现定量、可逆和大规模多重基因敲除。将MEGA应用于CAR T细胞耗竭模型，研究人员通过组合CRISPR筛选有力地抑制了抑制性受体的上调，并揭示了T细胞功能的成对调节因子。
此外，研究人员还实施了MEGA的药物调节，以受体不依赖的方式控制CAR的激活。MEGA能够多重破坏免疫调节代谢途径，从而在体外和体内增强CAR T细胞的适应性和抗肿瘤活性。
总之，MEGA为癌症免疫疗法及其他领域的应用提供了一个多功能的合成工具包。
附：英文原文
Title: A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells
Author: Victor Tieu, Elena Sotillo, Jeremy R. Bjelajac, Crystal Chen, Meena Malipatlolla, Justin A. Guerrero, Peng Xu, Patrick J. Quinn, Chris Fisher, Dorota Klysz, Crystal L. Mackall, Lei S. Qi
Issue&Volume: 2024-02-21
Abstract: CRISPR technologies have begun to revolutionize T cell therapies; however, conventionalCRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope.To address these challenges, we developed multiplexed effector guide arrays (MEGA),a platform for programmable and scalable regulation of the T cell transcriptome usingthe RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative,reversible, and massively multiplexed gene knockdown in primary human T cells withouttargeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion,we robustly suppressed inhibitory receptor upregulation and uncovered paired regulatorsof T cell function through combinatorial CRISPR screening. We additionally implementeddruggable regulation of MEGA to control CAR activation in a receptor-independent manner.Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways toenhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapyand beyond.
DOI: 10.1016/j.cell.2024.01.035
Source: https://www.cell.com/cell/abstract/S0092-8674(24)00102-8