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J Control Release. 2015 Nov 10;217:337-44. doi: 10.1016/j.jconrel.2015.08.051. Epub 2015 Sep 3.

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

Author information

1
Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium.
2
Laboratory of General Biochemistry and Physical Pharmacy, Ghent Research Group on Nanomedicine, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
3
Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, 500 Technology Square, Cambridge, MA, USA.
4
Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium. Electronic address: niek.sanders@ugent.be.
5
Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, 500 Technology Square, Cambridge, MA, USA. Electronic address: kitada@mit.edu.

Abstract

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.

KEYWORDS:

5-methylcytidine (PubChem CID: 92918); Antiviral innate immunity; Gene therapy; Modified RNA; N(1)-methylpseudouridine (PubChem CID: 99543); Nucleobase modifications; Toll-like receptor; mRNA; pseudouridine (PubChem CID: 15047)

PMID:
26342664
DOI:
10.1016/j.jconrel.2015.08.051
[Indexed for MEDLINE]

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