Codon-dependent translation of N1-ethylpseudouridine-modified mRNA reduces innate immunity while preserving vaccine immunogenicity.

Messenger RNA (mRNA) vaccines are a versatile platform for rapid vaccine development; however, nucleoside modifications must be optimized to balance translational efficiency and innate immune activation. Here, we evaluated N1-ethylpseudouridine (Et1Ψ) as an alternative to the widely used N1-methylpseudouridine (m1Ψ). Et1Ψ-modified mRNA exhibited a distinct codon-dependent translational sensitivity, with increased UUU codon content leading to reduced protein expression. Importantly, synonymous codon optimization that eliminated UUU codons restored translation efficiency to levels comparable to those of m1Ψ-modified mRNA across multiple cell types. Despite this constraint, Et1Ψ consistently reduced innate immune activation in vitro and in vivo. Following codon optimization, Et1Ψ-modified mRNA supported robust protein expression comparable to that from m1Ψ-modified mRNA. When applied to vaccine antigens, including SARS-CoV-2 spike and tetravalent dengue envelope domain III constructs, Et1Ψ-modified mRNA elicited strong humoral and cellular immune responses, with neutralizing activity comparable to that of m1Ψ-modified mRNA. Collectively, these findings identify Et1Ψ as an alternative nucleoside modification that combines reduced innate immunogenicity with codon-aware sequence optimization, highlighting codon-modification interactions as a key design principle for mRNA therapeutics.