Designing 5' UTR sequences improves the capacity of mRNA therapeutics in preclinical models of aging and obesity.

The utility of messenger RNA (mRNA) as a therapeutic modality has been widely demonstrated with the containment of COVID-19, yet the decisions in sequence design in the untranslated regions (UTRs) remain largely unexplored, especially in preclinical models. Here, we focus on the 5' UTR of mRNA and discover sequences that improve therapeutic potential in mouse models of aging and obesity. Bioinformatic analysis of RNA sequencing (RNA-seq), single-cell RNA-seq, ribosome profiling, and crosslinking and immunoprecipitation followed by sequencing data revealed that ribosomal protein (RP) mRNAs are abundant and ubiquitous but undergo distinct translational regulation by LARP1 and LARP4. Of 11 RP mRNAs, we find that the 5' UTRs of RPL18, RPL35, and RPS9 improve the protein output of synthetic mRNAs in human and mouse cells. Investigation of mutant 5' UTRs indicates that this improvement is independent of its terminal oligopyrimidine motif but strong in cells with high levels of reactive oxygen species. In aged mice and mice receiving a high-fat diet, synthetic mRNAs with the 5' UTR of RPS9 resulted in improved protein expression and enhanced humoral immunity through T helper cell 2 cytokines when encoding viral antigens. Altogether, our results highlight the importance of UTR sequence in expanding the therapeutic potential of synthetic mRNAs for aged individuals and those diagnosed with obesity.