Department of Electrical and Computer Engineering, North Dakota State University, Fargo, North Dakota, USA.
Biomedical Engineering Program, North Dakota State University, Fargo, North Dakota, USA.
Lab Chip. 2021 Sep 28;21(19):3748-3761. doi: 10.1039/d1lc00219h.
There is a growing interest for viral vector-free chimeric antigen receptor (CAR) T-cells due to its ability to kill cancer cells without adverse side effects. A potential avenue for manufacturing viral-vector free CAR T-cells is to utilize mRNA electroporation. One of the major concerns with mRNA electroporated CAR T-cells is the shorter cytotoxic lifespan of a few days, which is insufficient or not ideal for therapy. To better understand this issue and develop a potential solution, this study focused on examining the translation of electroporated mRNA to CAR molecules, time dependent degradation of CAR molecules and cytotoxicity produced by CAR T-cells on cancer cells. It was found that the initial expression of CAR molecules dictates the cytotoxicity. Initial CAR expression could be controlled by the experimental parameters such as electroporation time and mRNA concentration in the electroporation buffer. Experiments were carried out using a novel two-step electroporation that allows for controlled and uniform transfection of T-cells. These technical advancements and subsequent findings could provide a viable path for producing CAR T-cells with longer cytotoxic lifespans.
由于其能够在没有不良反应的情况下杀死癌细胞，因此人们对无病毒载体嵌合抗原受体 (CAR) T 细胞越来越感兴趣。制造无病毒载体 CAR T 细胞的一种潜在方法是利用 mRNA 电穿孔。mRNA 电穿孔 CAR T 细胞的一个主要问题是其细胞毒性寿命较短，仅有几天，这对于治疗来说是不够的或不理想的。为了更好地理解这个问题并开发潜在的解决方案，本研究专注于研究电穿孔 mRNA 到 CAR 分子的翻译、CAR 分子的时间依赖性降解以及 CAR T 细胞对癌细胞的细胞毒性。结果发现，CAR 分子的初始表达决定了细胞毒性。初始 CAR 表达可以通过实验参数来控制，如电穿孔时间和电穿孔缓冲液中的 mRNA 浓度。实验使用了一种新的两步电穿孔方法，可实现 T 细胞的受控和均匀转染。这些技术进步和随后的发现为生产具有更长细胞毒性寿命的 CAR T 细胞提供了可行的途径。
