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Article

Modulating the effectiveness of mRNA therapies using modified ribonucleotides

Modified ribonucleotides can boost the safety of mRNA vaccines and therapeutics


In vitro transcribed mRNA has become an effective and scalable therapeutic modality for a wide range of disease indications after the successful development of two COVID-19 vaccines.1 Vaccines and therapeutics based on mRNA technology work by instructing the body to make the protein of interest (e.g., a viral subunit in the case of a vaccine). However, the product works effectively only if the injected mRNA does not generate immunotoxicity.

This is where modified ribonucleotides can boost the safety of mRNA vaccines and therapeutics. The first generation of approved COVID-19 vaccines developed by Pfizer-BioNTech and Moderna Therapeutics (comirnaty® and spikevax®, respectively) are both based on mRNA containing the modified nucleotide N1-methyl-pseudouridine.2

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Immunogenicity of mRNA

As many viruses contain nucleic acids, cells have a defense system of pattern recognition receptors (PRRs) that sense foreign nucleic acids and trigger an innate immune response by the induction of cytokines. These PRRs include the endosomal receptors TLR3, TLR7, and TLR8, and the cytosolic receptors RIG-I and MDA-5. The first three recognize double and single-stranded RNA, while the latter two recognize 5′-triphosphate-modified RNA in addition to double-stranded RNA.3


In vitro transcribed and exogenously delivered mRNA is intrinsically immunogenic and can cause reduced translation.4,5 Induction of an immune response can, in principle, be a positive stimulus for a vaccine. However, an uncontrolled immune activation can compromise safe application. Additionally, immune signalling can prevent sufficient protein translation, thereby reducing the efficacy of a vaccine or therapeutic.5 Modifying mRNA structural elements, coding region, and polyadenylation can reduce immunogenicity as well as increase stability and translation, to a certain degree.6,7

mRNA IVT in vitro synthesis

Modified Ribonucleotides

Excessive mRNA immunogenicity can be prevented by incorporating modified ribonucleotides.1,2 Chemical modifications of mRNA are fairly common in nature and they influence post-transcriptional gene regulation in eukaryotes. More than 140 RNA modifications have been discovered across different organisms. Of those, pseudouridine (Ψ), N6-methyladenosine (m6A), 5-methylcytidine (m5C), and 2'-O-methylation (2′OMe) are found in mammalian mRNA.8

Use of modified ribonucleotides in mRNA production contributes to immunogenicity reduction, as well as increased protein production.5,9,10 Although the exact mechanisms by which modified ribonucleotides influence mRNA immunogenicity or protein production have not been defined, they exert their influence by altering one or more of the following properties of mRNA:

 

Reduced mRNA immunogenicity:

  • Altering RNA recognition  by intracellular receptors of the immune defense system5
  • Altering mRNA secondary structure and thereby influencing RNA-receptor interactions11,12
  • Altering antisense transcript synthesis by decreasing the “self-priming” effect of RNA polymerase, which reduces dsRNA13

 

Increased protein production: 

  • By regulating functional mRNA half-life14 
  • Via increased translation15,16

Production of modified mRNA via in vitro transcription

Production of mRNA vaccines and therapeutics is based on the in vitro transcription (IVT) reaction. The RNA polymerases used for the IVT reaction can also incorporate modified ribonucleotides that do not significantly alter base pairing.10 When a modified ribonucleotide is used in an IVT reaction, the regular nucleotide is replaced completely in the reaction mixture. Given that this results in a 100% substitution, use of a modified ribonucleotide must be compatible with all functional elements found in an mRNA transcript.17

mRNA workflow

Choose fit-for-purpose modified ribonucleotides to enhance your mRNA technology platform

Use modified ribonucleotides from Roche CustomBiotech in the synthesis of mRNA for therapeutics or vaccines. As high-performance reagents for the manufacture of therapeutics, our modified ribonucleotides are designed to meet quality and manufacturing specifications of the biopharmaceutical industry (fit-for-purpose). They are:

  • animal-origin-free (AOF)
  • free of ß-Lactam-antibiotics
  • extensively tested for impurities
  • validated via stringent QC release and cleaning methods 
  • supplied with supplementary documents for regulatory submissions

Meet Roche CustomBiotech experts from development and production

Discover CustomBiotech modified ribonucleotides and talk to our team about your needs in mRNA raw materials for your manufacturing process.
Contact us

Regulatory disclaimers are listed on the respective product pages.

References

  1. Modified uridines are the key to a successful message. Karikó K. Nat Rev Immunol. 2021 Oct;21(10):619. doi: 10.1038/s41577-021-00608-w.
  2. The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines. Morais P, Adachi H, Yu YT. Front Cell Dev Biol. 2021 Nov 4;9:789427. doi: 10.3389/fcell.2021.789427.PMID: 34805188
  3. Intracellular toll-like receptors.Blasius AL, Beutler B. Immunity. 2010 Mar 26;32(3):305-15. doi: 10.1016/j.immuni.2010.03.012.
  4. An origin of the immunogenicity of in vitro transcribed RNA. Mu X, Greenwald E, Ahmad S, Hur S. Nucleic Acids Res. 2018 Jun 1;46(10):5239-5249. doi: 10.1093/nar/gky177. PMID: 29534222
  5. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation.Anderson B. R., Muramatsu H., Nallagatla S. R., Bevilacqua P. C., Sansing L. H., Weissman D., Karikó K.  Nucleic Acids Res . 2010 Sep;38(17):5884-92. doi: 10.1093/nar/gkq347. Epub 2010 May 10.PMID: 20457754 PMCID: PMC2943593
  6. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Nahum S, Alan G H.  Cell. 2009 Feb 20;136(4):731-45. doi: 10.1016/j.cell.2009.01.042. PMID: 19239892 PMCID: PMC3610329
  7. Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals.Thess A, Grund S, Mui BL, Hope MJ, Baumhof P, Fotin-Mleczek M, Schlake T.Mol Ther. 2015 Sep;23(9):1456-64. doi: 10.1038/mt.2015.103.  PMID: 26050989
  8. Chemical modifications in the life of an mRNA transcript. Nachtergaele, S. & He, C.  Annu Rev Genet. 2018 Nov 23;52:349-372. PMID: 30230927 PMCID: PMC6436393 DOI: 10.1146/annurev-genet-120417-031522
  9. RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signalling. Durbin AF, Wang C, Marcotrigiano J, Gehrke L. mBio. 2016 Sep 20;7(5):e00833-16. doi: 10.1128/mBio.00833-16. PMID: 27651356
  10.  Effects of Chemically Modified Messenger RNA on Protein Expression. Li B, Luo X, Dong Y. Bioconjug Chem. 2016 Mar 16;27(3):849-53. doi: 10.1021/acs.bioconjchem.6b00090. PMID: 26906521
  11.  RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signalling. Durbin AF, Wang C, Marcotrigiano J, Gehrke L. mBio. 2016 Sep 20;7(5):e00833-16. doi: 10.1128/mBio.00833-16. PMID: 27651356
  12.  Suppression of RNA Recognition by Toll-like Receptors:The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Karikó K.; Buckstein M.; Ni H.; Weissman D. Immunity 2005, 23 (2), 165–175. 10.1016/j.immuni.2005.06.008
  13. Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Vaidyanathan S, Azizian KT, Haque AKMA, Henderson JM, Hendel A, Shore S, Antony JS, Hogrefe RI, Kormann MSD, Porteus MH, McCaffrey AP. Mol Ther Nucleic Acids. 2018 Sep 7;12:530-542. doi: 10.1016/j.omtn.2018.06.010. PMID: 30195789
  14. mRNA structure regulates protein expression through changes in functional half-life. Mauger DM, Cabral BJ, Presnyak V, Su SV, Reid DW, Goodman B, Link K, Khatwani N, Reynders J, Moore MJ, McFadyen IJ. Proc Natl Acad Sci U S A. 2019 Nov 26;116(48):24075-24083. doi: 10.1073/pnas.1908052116.PMID: 31712433
  15. Effects of Chemically Modified Messenger RNA on Protein Expression. Li B, Luo X, Dong Y. Bioconjug Chem. 2016 Mar 16;27(3):849-53. doi: 10.1021/acs.bioconjchem.6b00090. PMID: 26906521
  16. N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density. Svitkin YV, et al. Nucleic Acids Res. 2017. PMID: 28334758
  17. Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines. Nance KD, Meier JL. ACS Cent Sci. 2021 May 26;7(5):748-756. doi: 10.1021/acscentsci.1c00197.PMID: 34075344

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