One-step generation of modular CAR-T cells with AAV-Cpf1
One-step generation of modular CAR-T cells with AAV-Cpf1
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DOI：
10.1038/s41592-019-0329-7
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发表时间：
2019-03-01
期刊：
影响因子：
48
通讯作者：
Chen, Sidi
中科院分区：
文献类型：
--
作者：
Dai, Xiaoyun;Park, Jonathan J.;Chen, Sidi
免疫细胞工程为基础免疫学研究和免疫治疗开辟了新的能力。我们开发了一种系统，可有效生成嵌合抗原受体 (CAR) 工程 T 细胞 (CAR-T 细胞)，该系统通过简化的基因组工程显着增强了功能。通过利用反式激活 CRISPR（成簇规则间隔短回文重复序列）RNA (tracrRNA) 独立的 CRISPR-Cpf1 系统和腺相关病毒 (AAV)，我们能够一步高效地构建具有同源定向修复敲入和免疫检查点敲除的稳定 CAR-T 细胞（KIKO CAR-T 细胞）。 AAV-Cpf1 KIKO 系统的模块化能够在同一 T 细胞中灵活高效地生成两种不同 CAR 的双敲入。与基于Cas9的方法相比，AAV-Cpf1系统更有效地生成双敲入CAR-T细胞。 CD22特异性AAV-Cpf1 KIKO CAR-T细胞在细胞因子产生和癌细胞杀伤方面具有与Cas9 CAR-T细胞相当的效力，同时表达较低水平的耗竭标记物。这种多功能系统以简单和精确的方式开启了 T 细胞工程的新功能。
Immune-cell engineering opens new capabilities for fundamental immunology research and immunotherapy. We developed a system for efficient generation of chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) with considerably enhanced features by streamlined genome engineering. By leveraging trans-activating CRISPR (clustered regularly interspaced short palindromic repeats) RNA (tracrRNA)-independent CRISPR-Cpf1 systems with adeno-associated virus (AAV), we were able to build a stable CAR-T cell with homology-directed-repair knock-in and immune-checkpoint knockout (KIKO CAR-T cell) at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knock-in of two different CARs in the same T cell. Compared with Cas9-based methods, the AAV-Cpf1 system generates double-knock-in CAR-T cells more efficiently. CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to that of Cas9 CAR-T cells in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T-cell engineering with simplicity and precision.