Sub-stoichiometric 5-methoxyuridine modification enables tunable immune evasion and protein expression from synthetic...

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.