Nature Biotechnology ( IF 33.1 ) Pub Date : 2023-01-26 , DOI: 10.1038/s41587-022-01639-x
Xiaoyun Dai 1, 2, 3 , Jonathan J Park 1, 2, 3, 4, 5 , Yaying Du 1, 2, 3, 6 , Zhenkun Na 7, 8 , Stanley Z Lam 1, 2, 3 , Ryan D Chow 1, 2, 3, 4, 5 , Paul A Renauer 1, 2, 3, 5 , Jianlei Gu 9 , Shan Xin 1, 2, 3 , Zhiyuan Chu 1, 2, 3, 10 , Cun Liao 1, 2, 3, 11 , Paul Clark 1, 2, 3 , Hongyu Zhao 1, 9, 12, 13 , Sarah Slavoff 7, 8, 14 , Sidi Chen 1, 2, 3, 4, 5, 10, 13, 15, 16, 17, 18, 19, 20
|
The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.
中文翻译:

人类 T 细胞的大规模平行敲入工程
细胞治疗应用中的靶向敲入效率通常较低,且规模有限。在这项研究中,我们开发了 CLASH,这是一种实现高效、高通量敲入工程的系统。在 CLASH 中,Cas12a/Cpf1 mRNA 与混合的腺相关病毒结合,使用大规模平行同源定向修复介导同步基因编辑和精确转基因敲入,从而产生一组稳定整合的突变变体,每个变体都具有靶向基因编辑。我们将这项技术应用于原代人类 T 细胞,并使用 CD3、CD8 和 CD4 T 细胞在血癌和实体瘤模型中进行了时间进程 CLASH 实验,实现了有利 CAR-T 变体的混合生成和无偏选择。从 CLASH 实验中出现的独特 CRISPR RNA (crRNA) 在 CAR-T 中产生 PRDM1 的外显子 3 跳跃突变体,这导致这些细胞的增殖、干细胞样特性、中枢记忆和寿命增加,从而在包括实体瘤模型在内的多种癌症模型中具有更高的体内疗效。CLASH 的多功能性使其广泛适用于各种细胞和治疗工程应用。