一种经microRNA敲低PD-1的新型慢病毒载体在CAR-T细胞中的应用

Construction of a novel lentiviral vector knocking down PD-1 via microRNA and its application in CAR-T cells

摘要:

本研究将microRNA插入EF1α启动子的内含子中，构建携带沉默PD-1基因的miRNA的新型慢病毒载体，并将其应用于CAR-T细胞。通过流式细胞术检测慢病毒载体转导效率和PD-1沉默效率；Western blotting检测PD-1蛋白表达差异；荧光定量PCR检测microRNA相对表达情况；荧光素酶生物发光法和流式细胞术检测CAR-T细胞的能力。结果显示与U6转录microRNA的载体相比较，将microRNA插入到EF1-α内含子中的病毒载体转导效率更显著，对PD-1的敲低效率均达90%以上，且Western blotting结果验证了PD-1的敲低效果。另外通过荧光定量PCR，可显示出转导该新型慢病毒载体的Jurkat细胞内microRNA的相对表达量。荧光素酶生物发光法证实了CAR-T细胞针对靶细胞的特异杀伤性，流式细胞术结果表明沉默PD-1的CAR-T细胞相较于正常CAR-T细胞显示出更强的特异性杀伤能力。本研究成功构建了经microRNA敲低PD-1的新型慢病毒载体并验证了其转导效率的优越性，以及基于此载体表达的microRNA可高效地沉默PD-1；且应用此载体的CAR-T细胞能发挥更强的杀伤活性，从而为后续该CAR-T细胞治疗表达PD-L1的肿瘤奠定基础。

Abstract:

By inserting microRNAs into the intron of EF1α promoter, we constructed a novel lentiviral vector knocking down PD-1 gene via microRNA and applied it to CAR-T cells. Lentiviral transduction efficiency and PD-1-silencing efficiency were detected by flow cytometry. PD-1 expression was detected by Western blotting. Relative expression of microRNA was measured by Q-PCR. Cytotoxicity of CAR-T cells based on this vector was tested by luciferase bioluminescence and flow cytometry. Compared with lentiviral vector with microRNA transcribed by U6 promotor, the transduction efficiency of lentiviral vector with microRNA which was inserted into the intron of EF1α promoter was more significant, and the knockdown rate of PD-1 was more than 90%, which was validated by flow cytometry and Western blotting. And the relative expression level of microRNA in Jurkat cells transduced with this novel lentiviral vector was shown by Q-PCR. Compared with normal CAR-T cells, CAR-T cells based on this vector showed stronger cytotoxicity against PD-L1 positive Raji cells. We successfully constructed a novel lentiviral vector that knocked down PD-1 via microRNA and verified the superiority of its transduction efficiency and knockdown efficiency of PD-1. CAR-T cells based on this vector can exert a more powerful cytotoxicity, thus providing theoretical support for the subsequent treatment of PD-L1 positive tumors.