Codon optimality modulates cellular stress and innate immune responses triggered by exogenous RNAs

Despite advancements in RNA sequence design, evidence regarding the preferential use of synonymous codons on cellular stress and innate immune responses is lacking. To this end, we developed a new codon optimality formula to re-engineer the coding sequences of three luciferase reporters. We demonstrate that mRNAs enriched in optimal codons elicited dramatic increases in luciferase activities compared to less optimal sequences both in vitro and in vivo. Notably, transfecting low optimality test RNA suppresses the translation of co-transfected control mRNAs in dual reporter assays. Further, low optimality mRNAs activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2, a central event of the integrated stress response. eIF2 phosphorylation was suppressed by a GCN2 inhibitor, but not by other eIF2 kinase inhibitors. Using nucleoside-modified or circular RNAs also fully or partially abrogated these responses. Finally, optimal, but not non-optimal, circular RNAs have enhanced RNA lifespan and duration of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. Further, RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Together, we show that RNA coding sequence design is a key consideration for both mRNA and circular RNA therapeutics.