Engineered mRNA backbones for gene expression in human T cells

Current mRNA approaches in immuno-oncology lack specificity for optimal T cell mRNA expression, necessitating tailored mRNA expression systems. In this study, we developed novel mRNA constructs in which the standard α-globin (HBA1) 5' UTR is replaced with sequences derived from genes highly expressed in effector T cells. Using primary human T cells, expression levels of UTR-modified reporter genes were evaluated, revealing significant variability based on the substituted UTR. For instance, interferon gamma (IFN-γ) UTRs facilitated enhanced and sustained protein expression, whereas TNF UTRs showed diminished expression. Unexpectedly, the in silico-predicted RNA stability of the various UTR-modified constructs did not correlate with the altered expression. These UTR-mediated differences in protein expression were unique to T cells, as HEK cells introduced with the same constructs showed distinct expression profiles. CD19-CAR constructs expressed in T cells using various 5' UTRs demonstrated different protein expression and function toward antigen-positive target cells, as well as tonic signaling, manifested by the immune output in the absence of antigen. Specifically, for CD19-CAR, using the TIGIT 5' UTR proved optimal for achieving maximal reactivity while minimizing tonic signaling. These findings provide proof of concept for the pivotal role of T cell-specific UTRs in optimizing CAR-T cell functionality by fine-tuning expression, reducing tonic signaling, and minimizing off-target effects, thus emphasizing their potential in advancing the therapeutic potential of mRNA-based CAR-T cell therapies.