N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice

Messenger RNA as a therapeutic modality is becoming increasingly popular in the field of gene therapy. The realization that nucleobase modifications can greatly enhance the properties of mRNA by reducing the immunogenicity and increasing the stability of the RNA molecule (the Kariko paradigm) has be...

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Bibliographic Details
Published in:Journal of controlled release 2015-11, Vol.217, p.337-344
Main Authors: Andries, Oliwia, Mc Cafferty, Séan, De Smedt, Stefaan C, Weiss, Ron, Sanders, Niek N, Kitada, Tasuku
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cell Line, Tumor
Cell Survival
Cytidine - analogs & derivatives
Cytidine - chemistry
Humans
Luciferases, Firefly - genetics
Luciferases, Firefly - metabolism
Mice, Inbred BALB C
Pseudouridine - analogs & derivatives
Pseudouridine - chemistry
RNA, Messenger - chemistry
RNA, Messenger - pharmacology
Transfection
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Summary:Messenger RNA as a therapeutic modality is becoming increasingly popular in the field of gene therapy. The realization that nucleobase modifications can greatly enhance the properties of mRNA by reducing the immunogenicity and increasing the stability of the RNA molecule (the Kariko paradigm) has been pivotal for this revolution. Here we find that mRNAs containing the N(1)-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the current state-of-the-art pseudouridine (Ψ) and/or m5C/Ψ-modified mRNA platform by providing up to ~44-fold (when comparing double modified mRNAs) or ~13-fold (when comparing single modified mRNAs) higher reporter gene expression upon transfection into cell lines or mice, respectively. We show that (m5C/)m1Ψ-modified mRNA resulted in reduced intracellular innate immunogenicity and improved cellular viability compared to (m5C/)Ψ-modified mRNA upon in vitro transfection. The enhanced capability of (m5C/)m1Ψ-modified mRNA to express proteins may at least partially be due to the increased ability of the mRNA to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling. We believe that the (m5C/)m1Ψ-mRNA platform presented here may serve as a new standard in the field of modified mRNA-based therapeutics.
ISSN:1873-4995
DOI:10.1016/j.jconrel.2015.08.051