mRNA-based CAR T cell engineering: Unmodified mRNA enables high CAR expression without innate immune activation in T cells

Unmodified, uridine-containing mRNA is known to trigger antiviral immune responses, inflammatory signaling, and apoptosis in transfected cells. To avoid this and enable high expression, modified nucleosides such as N1-methylpseudouridine have become the gold standard for mRNA applications including T cell engineering, albeit at increased cost. Here, immune responses toward mRNA were evaluated across five primary human cell types. Remarkably, T cells, unlike other immune and non-immune cell types tested, exhibited no immune activation by unmodified mRNA. T cell viability and cytokine secretion remained unaffected, regardless of mRNA delivery method via lipid nanoparticles or electroporation. The absence of nucleotide modifications improved expression of chimeric antigen receptor (CAR) in activated T cells and CAR-T cell cytotoxic potency. By eliminating the need for mRNA-nucleoside modification in CAR-T cell engineering, our findings challenge existing paradigms and position mRNA as a non-inflammatory, minimally invasive and highly efficient tool for T cell engineering, while simplifying and reducing manufacturing cost.