Molecular Immunology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Department of Medical Biology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria, Australia.
J Leukoc Biol. 2020 Oct;108(4):1397-1408. doi: 10.1002/JLB.2MA0620-074R. Epub 2020 Jul 17.
The ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric Ag receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting NK cells have stalled in part by their resistance to traditional viral gene delivery systems. Here, an efficient approach is described to genetically edit human NK cells by electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes. Electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods are detailed. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, NCR1 and CISH are deleted in primary human NK cells and murine findings are validated on their key roles in regulating NK cell antitumor function.
利用病毒基因传递来对 CD8 T 细胞进行基因修饰，促进了新一代癌症免疫疗法的发展，例如嵌合抗原受体（CAR）T 细胞，这些细胞经过设计可特异性杀伤肿瘤细胞。靶向 NK 细胞的免疫疗法的发展在一定程度上受到其对传统病毒基因传递系统的抗性的阻碍。在这里，描述了一种通过电穿孔和 CRISPR-Cas9 核糖核蛋白（RNP）复合物来对人 NK 细胞进行基因编辑的有效方法。详细说明了电穿孔脉冲编码和缓冲液优化，以提高人 NK 细胞对蛋白质的摄取和细胞活力，并说明了这种方法相对于其他方法的效率。为了突出这项技术在 NK 细胞免疫疗法药物发现方面的变革性步骤，在原代人 NK 细胞中删除了 NCR1 和 CISH，并验证了它们在调节 NK 细胞抗肿瘤功能方面的关键作用。
