The transcriptional differences between mCAR-T and cmCAR-T cells

The study investigates the transcriptional differences between mCAR-T and cmCAR-T cells to understand their distinct cell-killing and cytokine release behaviors. RNA-seq experiments revealed that untransfected T cells, cmCAR-T, and mCAR-T cells exhibit significant transcriptomic differences on days 2 and 4. Notably, the GZMB gene, associated with cytotoxicity, was expressed at higher levels in cmCAR-T cells compared to mCAR-T cells. Analysis of gene signatures related to T cell activation and exhaustion showed distinct patterns between the two cell types. Differentially expressed genes (DEGs) highlighted enriched cytokine signaling in cmCAR-T cells on day 2 and interferon signaling on day 4. These findings suggest that while both CAR-T cell types have transient functional signals, cmCAR-T cells demonstrate enhanced and prolonged cytotoxicity compared to mCAR-T cells. Overall design: Control T cells (untransfected), Linear mRNA CAR-T or cmRNA CAR-T cells (n=3) were harvested at day 2 and day 4 post electroporation transfections. Subsequently, the total RNA was extracted from cell samples using TRIzol Reagent (Thermo Fisher Scientific, 15596026CN) for preparing RNA libraries using the VVAHTS® Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, NR605-0). Sequencing was performed using the Illumina NovaSeq 6000 platform.