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bioRxiv · 10.1101/2025.09.30.679448

Enhancement of mRNA translation efficiency through 5'-UTR engineering

Broset, E.·Blasco-Machin, I.·Lampaya, V.·Matute, C.·Toro-Cordova, A.·Martinez-Olivan, J.
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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.

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Broset, E., Blasco-Machin, I., Lampaya, V., Matute, C., Toro-Cordova, A., Martinez-Olivan, J.. 2025-10-01. Enhancement of mRNA translation efficiency through 5'-UTR engineering. https://doi.org/10.1101/2025.09.30.679448

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