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

Despite the widespread use of mRNA vaccines against COVID-19 little is known about how therapeutic RNAs are metabolized in vivo. Here, we have implemented nanopore sequencing to analyze individual therapeutic mRNA molecules, providing information about poly(A) tails. We show that Moderna mRNA-1273 vaccine is terminated by ~100 nucleotide long poly(A) tail followed by a m?Cm?AG sequence. In cell lines, mRNA-1273 is rapidly degraded in a process initiated by ?Cm?AG removal, followed by CCR4-NOT-mediated deadenylation. However, mRNA-1273 poly(A) tails can be extended by up to 200 nucleotides in more medically relevant preclinical models, particularly in macrophages. Re-adenylation, which increases the stability of mRNA-1273, is mediated by TENT5A poly(A) polymerase and is dependent on TENT5A attachment to the endoplasmic reticulum (ER), where these mRNAs are translated. Intriguingly, BioNTech/Pfizer BNT162b2 does not undergo as potent re-adenylation as mRNA-1273 despite the similarity and ER targeting. Notably, in vivo, TENT5A is expressed in immune cells that take up mRNA-1273 after intramuscular administration. Furthermore, TENT5A deficiency reduces antigen and specific immunoglobulin production post-immunization in mice. Overall, our findings provide an unexpected principle for enhancing the efficacy of therapeutic mRNAs,opening up new opportunities for improvement.