Bio and Drug Discovery Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, PO Box 107, Daejeon, 34114, Republic of Korea.
Medicinal Chemistry and Pharmacology, Korea University of Science and Technology, Daejeon, 34316, Republic of Korea.
Sci Rep. 2023 Jul 31;13(1):12365. doi: 10.1038/s41598-023-39597-2.
Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising modality for anti-cancer treatment. Its efficacy is quite remarkable in hematological tumors. Owing to their excellent clinical results, gene- modified cell therapies, including T cells, natural killer (NK) cells, and macrophages, are being actively studied in both academia and industry. However, the protocol to make CAR immune cells is too complicated, so it is still unclear how to efficiently produce the potent CAR immune cells. To manufacture effective CAR immune cells, we need to be aware of not only how to obtain highly infective viral particles, but also how to transduce CAR genes into immune cells. In this paper, we provide detailed information on spinoculation, which is one of the best known protocols to transduce genes into immune cells, in a methodological view. Our data indicate that gene transduction is significantly dependent on speed and duration of centrifugation, concentration and number of viral particles, the concentration of polybrene, and number of infected immune cells. In addition, we investigated on the optimal polyethylene glycol (PEG) solution to concentrate the viral supernatant and the optimized DNA ratios transfected into 293T cells to produce high titer of viral particles. This study provides useful information for practical production of the gene-modified immune cells using viral vectors.
嵌合抗原受体 (CAR) T 细胞疗法已成为癌症治疗的一种有前途的方法。它在血液肿瘤中的疗效非常显著。由于其出色的临床结果，包括 T 细胞、自然杀伤 (NK) 细胞和巨噬细胞在内的基因修饰细胞疗法正在学术界和工业界得到积极研究。然而，制造 CAR 免疫细胞的方案过于复杂，因此仍不清楚如何有效地产生有效的 CAR 免疫细胞。为了制造有效的 CAR 免疫细胞，我们不仅需要了解如何获得高感染力的病毒颗粒，还需要了解如何将 CAR 基因转导到免疫细胞中。在本文中，我们从方法学的角度详细介绍了一种众所周知的将基因转导到免疫细胞中的方法——转染。我们的数据表明，基因转导显著依赖于离心的速度和持续时间、病毒颗粒的浓度和数量、聚凝胺的浓度以及感染的免疫细胞的数量。此外，我们还研究了浓缩病毒上清液的最佳聚乙二醇 (PEG) 溶液以及转染 293T 细胞的优化 DNA 比例，以产生高滴度的病毒颗粒。这项研究为使用病毒载体实际生产基因修饰的免疫细胞提供了有用的信息。
