N1-Methylpseudouridine-incorporated mRNA provides enhanced protein expression and reduced immunogenicity

Therapeutic synthetic messenger RNAs (mRNAs) are now famous for developing vaccines against viruses and the treatment of cancers. The use of mRNA as a therapeutic has virtually no risk of genomic integration and mutagenesis of the host genome's critical regions. Synthetic mRNA used as a vaccine does not change the human (the host's) genome. Also, the gene expression platform based on mRNA does not need to enter the nucleus to function. Hence, mRNA can express a protein inside a cell, even in a cell that is not dividing. Because the expression of a protein from mRNA is transient, the mRNA platform is safer than plasmid RNA (pRNA) vector-based platforms. The idea of using mRNA as a protein replacement therapy is already over 20 years old. Since mRNAs are relatively labile and immunogenic, this approach did not become popular until more recently.

A study by Kariko's research group in 2005 demonstrated that the incorporation of base modifications found in natural RNAs into mRNA could reduce TLR mediated immunogenicity of mRNAs. The natural occurring nucleobases 5-methylcytidine (m5C), N6 -methyladenosine (m6A), pseudouridine (Ψ), 5-methyluridine (m5U), and 2-thiouridine (s2U) can be incorporated into mRNA either separately or in combination.

More recently, Andries et al. in 2015 tested if nucleobase modifications found in natural RNAs are superior to pseudouridine (Ψ) at enhancing the translational capacity of mRNA. The research group found that mRNAs containing the N¹-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the pseudouridine (Ψ) and/or m5C/Ψ-modified mRNAs.

The study observed an up to ~44-fold higher gene expression for double modified mRNAs or ~13-fold higher gene expression for single modified mRNAs upon cell line transfection. Modification of mRNA with (m5C/m1Ψ) resulted in reduced intracellular innate immunogenicity and improved cellular viability. The researchers suggest that this may be due to the mRNA's increased ability to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling.

 Figure 1: Chemical structures of modified nucleosides investigated in the studies.

Reference

Oliwia Andries, Séan Mc Cafferty, Stefaan C. De Smedt, Ron Weiss, Niek N. Sanders, Tasuku Kitada; N1-methyl-pseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. Journal of Controlled Release 217 (2015) 337–344. [PubMed]

K. Kariko, M. Buckstein, H. Ni, D. Weissman, Suppression of RNA recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23 (2005) 165–175. [PubMed]

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