Department of Rheumatology and Immunology, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China.
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Life Sciences, Yunnan University, Kunming, China.
Nat Biomed Eng. 2024 Feb;8(2):149-164. doi: 10.1038/s41551-023-01073-7. Epub 2023 Jul 27.
Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS-DNA interactions, affecting the production of the STING activator 2'3'-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.
病毒介导的 DNA 靶向重编程人类 T 细胞可导致随机基因组整合，电穿孔效率低且可能有毒性。本文中，我们发现原发性人 T 细胞中的电穿孔诱导毒性是由胞质途径 cGAS-STING（环磷酸鸟苷-腺苷酸（cGAMP）合酶-干扰素基因刺激物）介导的。我们还发现，通过筛选电穿孔条件鉴定出的等渗缓冲液可降低 cGAS-STING 监测，从而允许产生嵌合抗原受体（CAR）T 细胞，其 CAR T 细胞数量比标准电穿孔高 20 倍，并且在体内比慢病毒生成的 CAR T 细胞具有更高的抗肿瘤活性。电穿孔缓冲液的渗透压抑制了 cGAS-DNA 相互作用，影响了 STING 激活剂 2'3'-cGAMP 的产生。该缓冲液还可提高治疗相关原代 T 细胞和人造血干细胞的转染效率。我们的发现可能有助于优化电穿孔介导的 DNA 递送，以生产基因组工程 T 细胞。
