Department of Internal Medicine II, University Hospital of Würzburg, Würzburg, Germany.
Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany.
Methods Mol Biol. 2022;2521:41-66. doi: 10.1007/978-1-0716-2441-8_3.
Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Sleeping Beauty (SB) transposon vector components required for stable gene integration in human cells, with special emphasis on minicircle DNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cell engineering and describe assays that address transgene expression, biological potency and genomic vector copy numbers in polyclonal cell populations. Because transposons allow virus-free gene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities.
转染嵌合抗原受体（CAR）的人 T 淋巴细胞在某些血液系统癌症的基因和细胞免疫治疗中已被证明具有疗效和安全性。治疗性细胞产品需要合适的基因载体和基因工程方法，以确保其具有生物学效力，并且其制造具有经济可行性。转座子基因转移满足了这些需求，目前正在临床试验中进行评估。在本方案中，我们描述了在人类细胞中稳定基因整合所需的基本睡眠美人（SB）转座子载体组件，特别强调了微小环 DNA 载体和合成 mRNA 的使用。我们提供了一种基于荧光报告基因表达在培养的人细胞系上对载体组件进行功能验证的方案。最后，我们提供了 CAR-T 细胞工程的方案，并描述了用于检测多克隆细胞群体中转基因表达、生物效力和基因组载体拷贝数的检测方法。由于转座子允许使用裸露的核酸进行无病毒基因转移，因此任何配备生物安全水平 S1 设施的实验室都可以采用该方案。
