Why N1-methylpseudoUridine Modification is Selected for Marketed mRNA Vaccines
In early mRNA studies, mRNA entering cells would be recognized by the immune system as viral genetic material and eliminated. As a consequence, it's unable to perform the function of translating proteins. Even worse, it could induce severe immune responses and even lead to cell death.
Kariko and Weissman's work overcame the critical challenge. In the course of their collaboration, they noted that tRNA didn't trigger the excessive immune response due to the presence of pseudoUridine (Ψ) in the tRNA structure. Therefore, they tried to add pseudoUridine to the mRNA, and results suggested that the modified mRNA, after entering the cell, could be free from the attack of the immune system like tRNA, and translate specific proteins in the cell. In addition, they also found that pseudoUridine modification can improve the stability of mRNA and enhance its translation ability.
Based on these findings, Oliwia Andries et al. in 2015 found that the complete replacement of uridine with N1-methylpseudoUridine (m1Ψ) was more effective in reducing mRNA immunogenicity and enhancing mRNA protein expression ability than that of the complete replacement of uridine with pseudoUridine. In the global COVID-19 pandemic, two types of marketed COVID-19 mRNA vaccines, mRNA-1273 (Moderna) and BNT162b2 (Pfizer-Biontech), both adopted N1-methylfalse uridine triphosphate (m1ψTP) instead of UTP. It can be said that mRNA modification is crucial for the success of mRNA vaccines, and with the continuous expansion of the application of mRNA drugs, mRNA modification technology also needs to be continuously studied, understood, and optimized.
Much effort has been devoted in the past to understanding the mechanisms by which mRNA modifications inhibit innate immune responses and improve protein production, but relatively few studies have focused on their effects on the fidelity of protein synthesis. An article published in Cell Reports "N1-methylpseudoUridine found within COVID-19 mRNA vaccines produces faithful protein products" helps researchers go deeper into the mechanism of mRNA modification to better understand why N1-methylpseudoUridine modification rather than a pseudoUridine modification is used in all marketed mRNA vaccines.
The experimental results show that:
- m1Ψ does not affect the accuracy of the ribosome's selectivity.
- m1Ψ modified SARS-CoV-2 spike protein mRNA can be accurately translated by the ribosome.
- Ψ can be stably mismatched in the forming process of RNA double-stranded while m1Ψ cannot.
This article also systematically described the effects of the m1Ψ and Ψ on protein synthesis in an in vitro system and evaluated whether these effects were relevant to real-life applications, such as mRNA vaccines. The effect of nucleotide modification on the rate of peptide bond formation in the mRNA model was studied as well. It was found that m1Ψ reduced the overall rate of peptide bond formation in the bacterial recombination system.
However, the decrease of kpep didn't seem to affect the overall production of protein synthesis in eukaryotic cells or extracts, and it had even been found in many works of literature that the mRNA with m1Ψ can produce up to 10 times the protein. There are two possible mechanisms:
- m1Ψ-modified mRNA is more efficient in escaping toll-like receptors (TLRs);
- Modified-mRNA reduces the activation of integrated stress response mediated by PKR, thereby preventing the inhibition of translation initiation.
In conclusion, the m1Ψ has no obvious effect on decoding, and the m1Ψ-modified mRNA has superior in vivo characteristics. Therefore, the m1Ψ is more applicable in mRNA-based therapy.