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Archive > Volume 31 Issue 7 > 2024,31(7):655-661. DOI:10.3872/j.issn.1007-385X.2024.07.002 Prev Next
Basic Research
Expression of PD-1 shRNA enhances the killing ability of CD19-targeting CAR-T cells on tumor cells
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Abstract:
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.
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