The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1–6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

依赖病毒载体来生产用于过继性细胞疗法的基因工程免疫细胞仍然是转化瓶颈。在这里，我们报告了一种利用 DNA 修复途径同源介导的末端连接以及优化的试剂组成和递送的方法，用于 Cas9 诱导的大 DNA 有效负载有针对性地整合到原代人类 T 细胞中，毒性低，效率接近病毒载体（在多个目标基因组位点上以高达 70% 的速率定向敲入 1-6.7 kb 有效负载，并且细胞活力超过 80%）。我们使用该方法生产了带有工程化 T 细胞受体或嵌合抗原受体的 T 细胞，并表明这些细胞保持了低水平的耗竭标记物以及出色的增殖和细胞因子产生能力，并且它们在体外和体内引发了有效的抗肿瘤细胞毒性。老鼠。该方法很容易适应当前的良好生产实践和放大工艺，因此可以用作病毒载体的替代品，用于生产用于癌症免疫疗法的基因工程T细胞。