Optimizing mRNA translation efficiency through rational 5′UTR and 3′UTR combinatorial design

Advances in molecular medicine and biotechnology have demonstrated messenger RNA (mRNA)-based therapies to be a promising therapeutic modality for infectious diseases, genetic disorders, and cancers. However, key challenges persist, including low translation efficiency and short half-life of exogenous mRNA. The untranslated regions (UTRs) influence important parameters like mRNA stability and translation efficiency. This study adopted a combinatorial screening strategy to enhance exogenous mRNA translation efficiency by de novo designing 5′UTRs and combining them with multiple potential 3′UTRs. We designed a novel 5′UTR, 5UTR05, which exhibited comparable protein expression levels to the reference mRNA-1273 5′UTR that has been found to exhibit high expression in the COVID-19 vaccine development. Furthermore, the screening experiments found that combining 5UTR05 with both the 3′UTR of immunoglobulin heavy constant gamma 2 (IGHG2) and the 3′UTR of mitochondrially encoded 12S ribosomal RNA (mtRNR1) significantly improved mRNA translation efficiency, compared to individual 3′UTRs. Collectively, these findings provide valuable insights for UTR optimization strategies aimed at augmenting exogenous mRNA therapeutic translation. Continuing exploration of synergistic UTR combinations offers promise to advance customized mRNA constructs with optimized expression profiles tailored for diverse applications.