RT Journal Article SR Electronic T1 SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.12.01.518149 DO 10.1101/2022.12.01.518149 A1 Paweł S Krawczyk A1 Olga Gewartowska A1 Michał Mazur A1 Wiktoria Orzeł A1 Katarzyna Matylla-Kulińska A1 Sebastian Jeleń A1 Paweł Turowski A1 Tomasz Śpiewla A1 Bartosz Tarkowski A1 Agnieszka Tudek A1 Aleksandra Brouze A1 Aleksandra Wesołowska A1 Dominika Nowis A1 Jakub Gołąb A1 Joanna Kowalska A1 Jacek Jemielity A1 Andrzej Dziembowski A1 Seweryn Mroczek YR 2022 UL http://biorxiv.org/content/early/2022/12/01/2022.12.01.518149.abstract AB Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Moderna’s mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails.We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (mΨ), is terminated by a long poly(A) tail (∼100 nucleotides) followed by an mΨCmΨAG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of mΨCmΨAG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after mΨCmΨAG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after mΨCmΨAG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination.Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.Competing Interest StatementJacek Jemielity, Joanna Kowalska, Jakub Golab, and Dominika Nowis are founders of ExploRNA Therapeutics and Pawel Turowski is an employee of ExploRNA Therapeutics.