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 1×107 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 1×107 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 PPin vitro (PPConclusion: 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.

北京市双一流高层次人才科研启动经费（No. 9011451310032）