Molecular therapy. Nucleic acidsSep 5, 2026

Small amounts of 5-methoxyuridine in synthetic mRNAs help control immune response and protein production

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Sep 6, 2026

Abstract

Substituting 50% of uridines with 5-methoxyuridine (5moU) led to approximately 2-fold higher serum levels of protein compared to fully modified mRNA.

Sub-stoichiometric incorporation of 5moU may confer immune evasion while enhancing protein output.
Translation with 25% 5moU was comparable to fully modified mRNA in some cell types.
Using 50% 5moU maximized expression across all tested systems, often surpassing 100% fully modified mRNA.
5moU-modified mRNA showed reduced induction of interferon-stimulated genes compared to unmodified and fully modified mRNA.
5moU consistently suppressed innate immune responses to double-stranded RNA, unlike the fully modified version.

Simplified

mRNA therapeutics depend on nucleotide modification to evade innate immunity and sustain translation. Current clinical designs replace all uridine with N1-methylpseudouridine (m1Ψ). However, full substitution is costly and can perturb translational fidelity and RNA structure. Here, we show that sub-stoichiometric incorporation of 5-methoxyuridine (5moU) is sufficient to confer immune evasion while enhancing protein output. Substitution of 25% of uridines with 5moU supported translation comparable to fully m1Ψ-modified mRNA in some cell types, while 50% 5moU maximized expression across all systems tested, often outperforming 100% m1Ψ-modified mRNA. This effect was conserved in human primary cells and in vivo, where lipid nanoparticle-delivered mRNA encoding human IgG produced approximately 2-fold higher serum levels with 50% 5moU than with 100% m1Ψ. Transcriptomic profiling revealed reduced interferon-stimulated gene induction following delivery of 5moU-modified mRNA compared with unmodified and m1Ψ modified mRNA. Unlike m1Ψ, 5moU modification consistently suppressed innate responses to double-stranded RNA. Together, these data demonstrate that partial nucleotide substitution can outperform current mRNA modification strategies and expand the design space for therapeutic mRNAs.

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Small amounts of 5-methoxyuridine in synthetic mRNAs help control immune response and protein production

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