Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany.
Methods Mol Biol. 2022;2521:85-93. doi: 10.1007/978-1-0716-2441-8_5.
The efficient expression of T-cell receptors (TCRs) or chimeric antigen receptors (CARs) in primary human T cells is crucial for preclinical testing of receptor properties for adoptive T-cell therapies. Multiple streams of technological platforms have been developed in the recent decades to genetically modify primary T cells including nonviral platforms such as transposon-based systems (PiggyBac, Sleeping Beauty), TALENs, or CRISPR-Cas9). The production of CAR- or TCR-encoding retroviral vectors, however, is still the most commonly used technique both in preclinical as well as in clinical settings.In this chapter we describe a comprehensive 12-day protocol for (a) generating high-titered gamma-retroviral vector particles containing the transgene of interest (e.g., TCR , CAR ), (b) the isolation, activation and rapid expansion of primary T cells and (c) the stable genetic engineering of these T cells with the transgene for subsequent characterization.
高效表达 T 细胞受体 (TCRs) 或嵌合抗原受体 (CARs) 是原发性人 T 细胞进行受体特性的临床前测试的关键，用于过继性 T 细胞治疗。近几十年来，已经开发出多种技术平台来对原发性 T 细胞进行基因修饰，包括非病毒平台，如基于转座子的系统（PiggyBac、Sleeping Beauty）、TALENs 或 CRISPR-Cas9。然而，生产 CAR 或 TCR 编码的逆转录病毒载体仍然是最常用的技术，无论是在临床前还是临床环境中。在这一章中，我们描述了一个全面的 12 天方案，用于（a）生成含有感兴趣的转基因（例如 TCR、CAR）的高滴度γ-逆转录病毒载体颗粒，（b）原发性 T 细胞的分离、激活和快速扩增，以及（c）这些 T 细胞的稳定遗传工程化，以进行后续的表征。
