Enhancement of mRNA translation efficiency through 5'-UTR engineering

Abstract

The rapid progress of mRNA therapeutics has underscored a persistent challenge: achieving high protein expression at low dose. The 5 untranslated region (5-UTR) is a key regulator of translation initiation efficiency, prompting the question of whether a simple and portable modification could enhance expression across diverse designs. Here, we systematically engineered short repeats of the Kozak "core" motif (5-GCCACC-3) immediately upstream of the start codon and evaluated constructs incorporated into two widely used human UTRs (APO and HBB) in HeLa and HEK293T cells. Translation enhancement displayed a non-monotonic dependence on the number of Kozak repeats, with three repetitions consistently outperforming the native sequence and any other configuration. In mice, intramuscular lipid nanoparticle delivery of the three-copy design increased luciferase expression by [~]4-fold relative to the wild-type context and by up to [~]23-fold compared to a licensed vaccine UTR benchmark, providing clear in vivo relevance. These findings demonstrate that fine-tuning AUG-proximal Kozak elements constitutes a broadly applicable, UTR-independent strategy to enhance translation efficiency, offering a simple yet powerful principle for dose-sparing mRNA design in therapeutic applications.