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Modified uridines are the key to a successful message

In the 1990s, we started to investigate mRNA as a platform for protein replacement therapy. As these mRNAs encoded self-proteins, we did not think that mRNA transfection would generate any adverse immune effects. However, we found that transfecting human dendritic cells (DCs) with mRNA, or even with non-coding ribonucleotide homopolymers, induced inflammatory cytokines (Ni, H. et al., 2002).

At the time, we knew that DNA activates Toll-like receptor 9 (TLR9) and that double-stranded RNA can activate TLR3 and induce type I interferon. We hypothesized that one of the remaining TLR family members might sense single-stranded RNA. We also started to explore the activation of human DCs by different types of RNA to determine whether they all induce inflammatory cytokines. Natural RNAs are synthesized from the four basic nucleotides, but some of the nucleosides can be post-transcriptionally modified. We found that tRNA, which is known to be enriched in modified nucleosides, was non-inflammatory, and that TLR7 and TLR8 sense single-stranded RNA. We set out to generate RNA with modified nucleosides by in vitro synthesis. Surprisingly, the replacement of uridine with pseudouridine rendered the RNAs non-immunogenic (Karikó, K. et al., 2005).

In subsequent studies we demonstrated that mRNA containing pseudouridine was an ideal molecule for protein replacement therapy because it was efficiently translated and, unlike its unmodified counterpart, did not induce interferon in mice. Indeed, the injection of a small amount of mRNA was sufficient for the encoded protein to exert its therapeutic effect (Karikó, K. et al., 2008; Karikó, K. et al., 2012).

“the replacement of uridine with pseudouridine rendered the RNAs non-immunogenic”

In parallel to these studies, we investigated mRNA as a platform for vaccine development. We predicted that uridine-containing (and thereby self-adjuvanted) mRNA encoding viral antigens would be optimal for vaccine development. Amazingly, non-immunogenic mRNA containing modified uridines also turned out to be a more suitable molecule for vaccine development (Pardi et al., 2017). Indeed, the first mRNA-based vaccines to receive regulatory authorization — developed by Moderna and by BioNTech/Pfizer for COVID-19 — are both based on 1-methylpseudouridine-containing mRNA.


Original articles

  1. Ni, H. et al. Extracellular mRNA induces dendritic cell activation by stimulating tumor necrosis factor-alpha secretion and signaling through a nucleotide receptor. J. Biol. Chem. 277, 12689–12696 (2002)

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  2. Karikó, K. et al. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005)

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  3. Karikó, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008)

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  4. Karikó, K. et al. Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol. Ther. 20, 948–953 (2012)

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  5. Pardi, N. et al. Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination. Nature 543, 248–251 (2017)

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Correspondence to Katalin Karikó.

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Competing interests

K.K. is named on patents that describe the use of nucleoside-modified mRNA as a platform to deliver therapeutic proteins. She is employee of BioNTech SE, a company that develops mRNA-based therapies.

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Karikó, K. Modified uridines are the key to a successful message. Nat Rev Immunol 21, 619 (2021).

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