The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double-stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator-like effector nuclease (TALEN)-mediated gene editing in primary T cells to improve nonviral gene knock-in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single-stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time-controlled two-step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T-cell receptor alpha constant region (TRAC) locus to generate ‘off-the shelf’ CAR-T cells.

中文翻译：

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优化的两步电穿孔过程，以实现有效的非病毒介导的基因插入原代 T 细胞

过去几年基因编辑技术的发展使得转基因能够精确有效地插入到各种细胞类型的基因组中。使用同源定向修复和设计核酸酶的敲入方法通常依赖于病毒载体，这会极大地影响基因编辑治疗产品的制造成本和时间表。一个有吸引力的替代方案是使用裸 DNA 作为修复模板。然而，这种策略面临着挑战，例如从双链 DNA (dsDNA) 到原代细胞的细胞毒性。在这里，我们试图研究原代 T 细胞中转录激活因子样效应核酸酶 (TALEN) 介导的基因编辑的动力学，以改善非病毒基因敲入。利用这些知识，我们开发了一种基于短单链寡核苷酸或大 (2 Kb) 线性裸 dsDNA 序列的快速有效的基因插入策略。我们证明了时间控制的两步转染方案可以显着提高非病毒转基因在原代 T 细胞中的整合效率。使用这种方法，当在 T 细胞受体 α 恒定区插入嵌合抗原受体 (CAR) 时，我们实现了高达 30% 的 T 细胞修饰。TRAC ) 基因座生成“现成的”CAR-T 细胞。