N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting

Thomas E. Mulroney, Tuija Pöyry, Juan Carlos Yam-Puc, Maria Rust, Robert F. Harvey, Lajos Kalmar, Emily Horner, Lucy Booth, Alexander P. Ferreira, Mark Stoneley, Ritwick Sawarkar, Alexander J. Mentzer, Kathryn S. Lilley, C. Mark Smales, Tobias Haar, Lance Turtle, Susanna Dunachie, Paul Klenerman, James E. D. Thaventhiran () and Anne E. Willis ()
Additional contact information
Thomas E. Mulroney: University of Cambridge
Tuija Pöyry: University of Cambridge
Juan Carlos Yam-Puc: University of Cambridge
Maria Rust: University of Cambridge
Robert F. Harvey: University of Cambridge
Lajos Kalmar: University of Cambridge
Emily Horner: University of Cambridge
Lucy Booth: University of Cambridge
Alexander P. Ferreira: University of Cambridge
Mark Stoneley: University of Cambridge
Ritwick Sawarkar: University of Cambridge
Alexander J. Mentzer: University of Oxford
Kathryn S. Lilley: University of Cambridge
C. Mark Smales: University of Kent
Tobias Haar: University of Kent
Lance Turtle: University of Liverpool
Susanna Dunachie: Oxford University Hospitals NHS Foundation Trust
Paul Klenerman: Oxford University Hospitals NHS Foundation Trust
James E. D. Thaventhiran: University of Cambridge
Anne E. Willis: University of Cambridge

Nature, 2024, vol. 625, issue 7993, 189-194

Abstract: Abstract In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3–5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.

Date: 2024
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DOI: 10.1038/s41586-023-06800-3

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