‌RNA secondary structure and qRT-PCR analyses pertained to expressed anti-CD25 CAR in NK-92 cell line

Background and Objectives: Tumor-infiltrating regulatory T (TI-Treg) cells perform the significant function in cancer immune escape. In this study, the third generation CAR construct was designed against human CD25 antigen, the significant cell surface biomarker of TI-Tregs. Methods: Initially, the construct of anti-CD25 CAR was designed. Using RNAfold web server, the RNA secondary structure was evaluated. Also, utilizing lentiviral vectors, NK-92 cell line was transduced. Afterward, the expression level of anti-CD25 CAR RNA was assessed by qRT-PCR in NK-92 cells transduced with CAR and mock transfer vectors and also untreated cells. Results: The RNA secondary structure was stable. Also, the expression level of anti-CD25 CAR RNA in transduced NK-92 cells by pCDH-513B-1-anti-CD25 CAR transfer vector was significantly higher than transduced NK-92 cells by mock transfer vector and untreated cells (p˂0.0001). Conclusion: The present study on anti-CD25 CAR RNA showed that this type of CAR transcripts were stable and expressed at high level. In fact, this type of CAR can be further studied in the future as a tool to remove the cancer immune escape in all types of solid and liquid cancers.