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

The COVID-19 mRNA vaccines demonstrated the power of mRNA medicines. Despite advancements in 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 proprietary codon optimality matrix to re-engineer the coding sequences of three luciferase reporters. We demonstrate that optimal mRNAs elicited dramatic increases in luciferase activities compared to non-optimal sequences. Notably, transfecting an optimal RNA affects the translation of other RNAs in the cell including control transcripts in dual luciferase assays. This held true in multiple cell lines and for an unrelated reporter. Further, non-optimal mRNAs preferentially activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2α, a central event of the integrated stress response. Using nucleoside-modified or circular RNAs partially or fully abrogated these responses. Finally, we show that circularizing RNAs enhances both RNA lifespan and durability of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. However, we also show that RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Hence, RNA sequence engineering, chemical, and topological modifications must all be combined to elicit favorable therapeutic outcomes.