Fig. 3

Scarless cmRNAs enhanced and prolonged the protein expression of the CD19 CAR on primary human T cells. a, b Structures of CAR-encoding cmRNA and second-generation anti-CD19 CAR (a) or anti-GPRC5D CAR (b). c, d Primary human T cells were activated and transfected with linear mRNAs (blue), scarred cmRNAs (green) or scarless cmRNAs (red) on day 4. Representative histograms of CAR expression analysis from 6 h to 7 days after transfection. PE-labeled human CD19 (20-291) protein was used for anti-CD19 CAR detection, MonoRab^TM Rabbits Anti-scFv Cocktail (PE) was used for anti-GPRC5D CAR detection, and Zombie Violet^TM Dye was used to exclude dead cells. e, f Proportion of CAR-expressing cells indicated by anti-CD19 CAR⁺ cells (E) and anti-GPRC5D CAR⁺ cells (F) from 6 h to 7 days post-transfection (n = 3 independent experiments/donors). g, h The median intensity of CAR expression from 6 h to 7 days post-transfection (n = 3 independent experiments)