Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability

TLDR

In vitro‑transcribed mRNAs hold therapeutic promise but are limited by their instability and strong immunogenicity. The study examined whether incorporating naturally modified nucleotides, specifically pseudouridine, into mRNA transcripts could improve these biological properties. Pseudouridine‑modified mRNAs exhibited higher translational capacity, greater protein expression in the spleen, and markedly reduced immunogenicity, demonstrating that nucleoside modification enhances mRNA stability and translational efficiency while diminishing immune activation, making it a promising platform for gene therapy and vaccination.

Abstract

In vitro-transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, in its present form, mRNA is unfeasible for clinical use because of its labile and immunogenic nature. Here, we investigated whether incorporation of naturally modified nucleotides into transcripts would confer enhanced biological properties to mRNA. We found that mRNAs containing pseudouridines have a higher translational capacity than unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into mice at 0.015-0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the unmodified mRNA was immunogenic, inducing high serum levels of interferon-alpha (IFN-alpha). These findings indicate that nucleoside modification is an effective approach to enhance stability and translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties conferred by pseudouridine make such mRNA a promising tool for both gene replacement and vaccination.