mRNA-based CAR T-cells manufactured by miniaturized two-step electroporation produce selective cytotoxicity toward target cancer cells,Lab on a Chip

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mRNA-based CAR T-cells manufactured by miniaturized two-step electroporation produce selective cytotoxicity toward target cancer cells
Lab on a Chip ( IF 6.1 ) Pub Date : 2021-08-09 , DOI: 10.1039/d1lc00219h
Vidura Jayasooriya ₁ , Beth Ringwelski ₂ , Glenn Dorsam ₃ , Dharmakeerthi Nawarathna _{1,

2}

Affiliation

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.

中文翻译：

通过微型两步电穿孔制造的基于 mRNA 的 CAR T 细胞对靶癌细胞产生选择性细胞毒性

人们对无病毒载体嵌合抗原受体 (CAR) T 细胞的兴趣日益浓厚，因为它能够杀死癌细胞而没有不良副作用。制造无病毒载体的 CAR T 细胞的一个潜在途径是利用 mRNA 电穿孔。mRNA 电穿孔 CAR T 细胞的主要问题之一是几天的细胞毒性寿命较短，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，本研究的重点是检查电穿孔 mRNA 向 CAR 分子的翻译、CAR 分子的时间依赖性降解以及 CAR T 细胞对癌细胞产生的细胞毒性。发现CAR分子的初始表达决定了细胞毒性。初始 CAR 表达可以通过电穿孔缓冲液中的电穿孔时间和 mRNA 浓度等实验参数来控制。使用允许控制和均匀转染 T 细胞的新型两步电穿孔进行实验。这些技术进步和随后的发现可以为生产具有更长细胞毒性寿命的 CAR T 细胞提供一条可行的途径。

更新日期：2021-08-13

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