SARS-CoV-2 mRNA vaccine is re-adenylatedin vivo, enhancing antigen production and immune response

Abstract

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 (m Psi), is terminated by a long poly(A) tail (~100 nucleotides) followed by an mpsimpsi AG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of mPsiPsiAG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after mPsimPsiAG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after mPsimPsiAG 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.

TENT5-mediated polyadenylation of mRNAs encoding secreted proteins is essential for gametogenesis in mice

“…Accordingly, our recent data unexpectedly indicates that the Moderna vaccine mRNA-1273, which encodes Spike antigen targeted to the ER, is an efficient substrate for TENT5 polymerases. The Moderna vaccine is completely synthetic and optimized for efficient translation rather than cytoplasmic polyadenylation 55 . This points to a simple model in which efficiently translated mRNA with a leader peptide that are recruited to the ER are polyadenylated by TENT5s, which are enriched at the surface of the ER 29,30,56 .…”

Polyadenylation of mRNAs encoding secreted proteins by TENT5 family of enzymes is essential for gametogenesis in mice

“…The same is for completely synthetic mRNA, such as the Moderna mRNA-1273 vaccine, which is taken by macrophages after intermuscular injection. Vaccine mRNA encoding coronavirus Spike protein is targeted to ER and recruits TENT5A for polyadenylation and stabilization 53 . Consequently, TENT5A is essential for efficient antigen production and immune responses.…”

Strategic Deactivation of mRNA COVID-19 Vaccines: New Applications for RIBOTACs and siRNA Therapy

“…Interestingly, Krawczyk et al describe that the Moderna mRNA-1273 has been engineered with a ψCψAG sequence following the long poly (A) tail and that this ψCψAG sequence is removed after intramuscular injection. Although removal of the ψCψAG sequence promotes mRNA-1273 de-adenylation and subsequent degradation in vitro, the injected mRNA-1273 becomes re-adenylated in vivo [30]. Furthermore, synthesized mRNA must be purified from reaction components in accordance with clinical quality standards.…”