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 RNAs suppress the translation of co-transfected control mRNAs in dual reporter assays. Transcriptomic profiling revealed temporally distinct waves of cellular stress, comprising an early ribotoxic stress response followed by a sustained integrated stress response and interferon-driven innate immune program. Cells responded to both dsRNA contaminants, which was mitigated by cellulose purification, and also rare codon-mediated ribosome stalling signal activating GCN2 independently of dsRNA. Translation suppression by low optimality mRNAs was driven by eIF2α phosphorylation, which was suppressed by a GCN2 inhibitor, but not by other eIF2α kinase inhibitors, and both cellulose purification and GCN2 inhibition each produced partial translational rescue. Using nucleoside-modified or circular RNAs also fully or partially abrogated these responses. Finally, only 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.