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

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 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.