表达PD-1 shRNA增强靶向CD19 CAR-T细胞对肿瘤细胞的杀伤能力  

Expression of PD-1 shRNA enhances the killing ability of CD19-targeting CAR-T cells on tumor cells

作　　者：林伟 朱晶晶 刘秀盈 王建勋 LIN Wei;ZHU Jingjing;LIU Xiuying;WANG Jianxun(School of Life Sciences,Beijing University of Traditional Chinese Medicine,Beijing 102400,China)

机构地区：[1]北京中医药大学生命科学学院,北京102400

出　　处：《中国肿瘤生物治疗杂志》2024年第7期655-661,共7页Chinese Journal of Cancer Biotherapy

基　　金：北京市双一流高层次人才科研启动经费(No.9011451310032)。

摘　　要：目的:设计和构建表达PD-1 shRNA的靶向CD19 CAR-T细胞并验证其体外肿瘤细胞杀伤能力。方法:设计并构建表达PD-1 shRNA的CD19 CAR分子基因,将其包装成逆转录病毒载体,通过qPCR法检测病毒载体拷贝数。将慢病毒转导人原代T细胞,获得三种CAR-T细胞,分别为RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞。采用qPCR法检测三种CAR-T细胞中PD-1 mRNA的表达水平,流式细胞术检测三种CAR-T细胞中PD-1表达水平,萤光素酶报告基因实验、流式细胞术检测在不同效靶比时CAR-T细胞对CD19阳性靶细胞(人淋巴瘤daudi细胞)的杀伤功能。结果:RNAU6-CD19 CAR、PD-1 shRNA1-CD19 CAR、PD-1 shRNA2-CD19 CAR三种CAR分子成功包装成逆转录病毒载体,病毒载体拷贝数均高于1×10^(7)拷贝/mL,转导人原代T细胞获得CAR-T细胞,RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞转导效率分别为43.1%、55.1%、41.7%。与RNAU6-CD19 CAR-T细胞相比,PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞中PD-1 mRNA表达水平均显著降低(均P<0.01)、细胞表面PD-1表达水平更低(均P<0.01)、体外对daudi细胞的杀伤率更高(P<0.05或P<0.01)。结论:成功构建表达PD-1 shRNA的靶向CD19 CAR-T细胞,其对CD19阳性靶细胞的杀伤率显著提高,PD-1 mRNA及其翻译产物PD-1的表达减少,CAR-T细胞的耗竭减缓。Objective:To design and construct CD19-targeting CAR-T cells expressing PD-1 shRNA and validate their anti-tumor function in vitro.Methods:The authors designed and constructed CD19 CAR molecule gene expressing PD-1 shRNA,and packaged them into retroviral vector using packaging cells.The viral vector copy number was detected by qPCR,and then human primary T cells were transduced to obtain CAR-T cells,which were divided into three groups:RNAU6-CD19 CAR-T,PD-1 shRNA1-CD19 CAR-T,and PD-1 shRNA2-CD19 CAR-T cells.qPCR was applied to detect the expression levels of PD-1 mRNA in three groups of CAR-T cells.Flow cytometry was used to detect the expression level of PD-1 on CAR-T cells in three groups.The luciferase reporter gene method and flow cytometry were used to detect the killing function of CAR-T cells against target cells(human lymphoma daudi cells)at different efficacy to target ratios.Results:Three groups of CAR molecules,namely RNAU6-CD19 CAR,PD-1 shRNA1-CD19 CAR and PD-1 shRNA2-CD19 CAR,were successfully packaged into retroviral vector,in which all retroviral vector copy numbers were higher than 1×10^(7) copies/mL.CAR-T cells were obtained by transducing human primary T cells.The CAR-T transduction efficiencies of RNAU6-CD19 CAR-T,PD-1 shRNA1-CD19 CAR-T and PD-1 shRNA2-CD19 CAR-T cells were 43.1%,55.1%,and 41.7%respectively.Compared with RNAU6-CD19 CAR-T cells,PD-1 shRNA1-CD19 CAR-T and PD-1 shRNA2-CD19 CAR-T cells showed a significant decrease in the expression level of PD-1 mRNA(all P<0.01),lower expression level of PD-1 on cell surface s(all P<0.01),and higher killing efficiency against CD19 positive target cells(daudi cells)in vitro(P<0.05 or P<0.01).Conclusion:Successfull construction of CD19-targeting CAR-T cells expressing PD-1 shRNA can improve the killing efficiency against CD19 positive target cells,reduce the expressions of PD-1 mRNA and its translation product PD-1,and slow down the depletion of CAR-T cells.

关 键 词：CD19 PD-1 嵌合抗原受体 T细胞 RNA干扰 

分 类 号：R730.51[医药卫生—肿瘤] R392.12[医药卫生—临床医学]