Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors

Chimeric antigen receptor engineered T cell (CAR-T) immunotherapy has shown efficacy against a subset of hematological malignancies1,2, yet its autologous nature and ineffectiveness against epithelial and solid cancers limit widespread application. To overcome these limitations, targeted nucleases have been used to disrupt checkpoint inhibitors and genes involved in alloreactivity3–6. However, the production of allogeneic, “off-the-shelf” T cells with enhanced function requires multiplex genome editing strategies that risk off-target effects, chromosomal rearrangements, and genotoxicity due to simultaneous double-strand break (DSB) induction at multiple loci7–10. Moreover, it has been well documented that DSBs are toxic lesions that can drive genetic instability11,12. Alternatively, CRISPR/Cas9 base editors afford programmable enzymatic nucleotide conversion at targeted loci without induction of DSBs13,14. We reasoned this technology could be used to knockout gene function in human T cells while minimizing safety concerns associated with current nuclease platforms. Through systematic reagent and dose optimization, we demonstrate highly efficient multiplex base editing and consequent protein knockout in primary human T cells at loci relevant to the generation of allogeneic CAR-T cells including the T cell receptor a constant (TRAC) locus, ß-2 microglobulin (B2M), and programmed cell death 1 (PDCD1). Multiplex base edited T cells equipped with a CD19 CAR killed target cells more efficiently; and importantly, both DSB induction and translocation frequency were greatly reduced compared to cells engineered with Cas9 nuclease. Collectively, our results establish a novel multiplex gene editing platform to enhance both the safety and efficacy of engineered T cell-based immunotherapies.

嵌合抗原受体工程化T细胞（Chimeric antigen receptor engineered T cell, CAR-T）免疫疗法已在部分血液系统恶性肿瘤中展现出治疗效果¹,²，然而其自体属性以及对上皮癌和实体瘤的治疗无效性，限制了其大规模应用。为克服上述局限，研究人员已利用靶向核酸酶破坏免疫检查点抑制剂及参与同种反应性的基因³–⁶。然而，制备功能增强的同种异体"off-the-shelf"T细胞需要多重基因组编辑策略，但这类策略因在多个基因座同时诱导双链断裂（double-strand break, DSB），存在脱靶效应、染色体重排及遗传毒性的风险⁷–¹⁰。此外，已有充分研究证实，双链断裂（DSB）是可引发遗传不稳定的毒性损伤¹¹,¹²。与之不同的是，CRISPR/Cas9碱基编辑器可在靶向基因座实现可编程的酶促核苷酸转换，且不会诱导双链断裂（DSB）¹³,¹⁴。我们推测，该技术可用于敲除人T细胞中的基因功能，同时最大限度降低当前核酸酶平台所带来的安全隐患。经过系统的试剂与剂量优化，我们在原代人T细胞中实现了高效的多重碱基编辑及后续的蛋白敲除，所靶向的基因座与同种异体CAR-T细胞的制备相关，包括T细胞受体α恒定区（T cell receptor α constant, TRAC）基因座、β-2微球蛋白（β-2 microglobulin, B2M）以及程序性细胞死亡蛋白1（programmed cell death 1, PDCD1）。搭载CD19嵌合抗原受体的多重碱基编辑T细胞可更高效地杀伤靶细胞；尤为重要的是，与使用Cas9核酸酶工程化改造的细胞相比，这类细胞的双链断裂诱导率与染色体易位频率均大幅降低。综上，本研究结果构建了一种新型多重基因编辑平台，可同时提升工程化T细胞免疫疗法的安全性与有效性。