Identification of novel 5' UTR variants for enhanced mRNA translation and vaccine immunogenicity.

mRNA-based therapeutics represent a highly promising platform for the prevention and treatment of diseases. However, further optimization is required to improve mRNA stability and translational efficiency. The 5' untranslated region (5' UTR) is a critical regulatory element that governs pre-initiation complex assembly and directly influences protein expression levels, making it an important target for rational design. The diverse challenges facing mRNA therapeutics necessitate the development of improved 5' UTR elements adaptable to specific applications. In this study, we evaluated the effects of five different 5' UTR variants, selected based on published data, on firefly luciferase (FLuc) production in cell culture and in an in vivo bioluminescence imaging model in animals. Subsequently, all variants were tested as components of mRNA vaccines against SARS-CoV-2. Our findings demonstrate that the H1.2-TISU and Synth 5' UTRs with mRNA constructs produce increased levels of reporter protein expression in mice, whereas mRNA constructs incorporating the H1.2-TISU 5' UTR demonstrated enhanced humoral immunogenicity compared to the 5' UTR derived from human alpha-globin (HBA) mRNA. Thus, the identified 5' UTRs (H1.2-TISU and Synth) represent promising, in vivo-validated candidates for incorporation into mRNA-based therapeutics and may substantially enhance their efficacy.