Biology:Nucleoside-modified messenger RNA

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Short description: Chemically modified messenger RNA

A nucleoside-modified messenger RNA (modRNA) is a synthetic messenger RNA (mRNA) in which some nucleosides are replaced by other naturally modified nucleosides or by synthetic nucleoside analogues.[1] modRNA is used to induce the production of a desired protein in certain cells. An important application is the development of mRNA vaccines, of which the first authorized were COVID-19 vaccines (such as Comirnaty and Spikevax).

Background

A ribosome (depicted in green) creates a protein (depicted here as a string of beads representing amino acids) encoded in an mRNA (depicted as a ribbon of nucleotides) that may be modified to reduce inflammation in the cell.

mRNA is produced by synthesising a ribonucleic acid (RNA) strand from nucleotide building blocks according to a deoxyribonucleic acid (DNA) template, a process that is called transcription.[2] When the building blocks provided to the RNA polymerase include non-standard nucleosides such as pseudouridine — instead of the standard adenosine, cytidine, guanosine, and uridine nucleosides — the resulting mRNA is described as nucleoside-modified.[3]

Production of protein begins with assembly of ribosomes on the mRNA, the latter then serving as a blueprint for the synthesis of proteins by specifying their amino acid sequence based on the genetic code in the process of protein biosynthesis called translation.[4]

Overview

To induce cells to make proteins that they do not normally produce, it is possible to introduce heterologous mRNA into the cytoplasm of the cell, bypassing the need for transcription. In other words, a blueprint for foreign proteins is "smuggled" into the cells. To achieve this goal, however, one must bypass cellular systems that prevent the penetration and translation of foreign mRNA. There are nearly-ubiquitous enzymes called ribonucleases (also called RNAses) that break down unprotected mRNA.[5] There are also intracellular barriers against foreign mRNA, such as innate immune system receptors, toll-like receptor (TLR) 7 and TLR8, located in endosomal membranes. RNA sensors like TLR7 and TLR8 can dramatically reduce protein synthesis in the cell, trigger release of cytokines such as interferon and TNF-alpha, and when sufficiently intense lead to programmed cell death.[6]

The inflammatory nature of exogenous RNA can be masked by modifying the nucleosides in mRNA.[7] For example, uridine can be replaced with a similar nucleoside such as pseudouridine (Ψ) or N1-methyl-pseudouridine (m1Ψ),[8] and cytosine can be replaced by 5-methylcytosine.[9] Some of these, such as pseudouridine and 5-methylcytosine, occur naturally in eukaryotes,[10] while m1Ψ occurs naturally in archaea.[11] Inclusion of these modified nucleosides alters the secondary structure of the mRNA, which can reduce recognition by the innate immune system while still allowing effective translation.[9]

Significance of untranslated regions

A normal mRNA starts and ends with sections that do not code for amino acids of the actual protein. These sequences at the 5′ and 3′ ends of an mRNA strand are called untranslated regions (UTRs). The two UTRs at their strand ends are essential for the stability of an mRNA and also of a modRNA as well as for the efficiency of translation, i.e. for the amount of protein produced. By selecting suitable UTRs during the synthesis of a modRNA, the production of the target protein in the target cells can be optimised.[5][12]

Delivery

Comparing uptake of RNA and modRNA by the cell

Various difficulties are involved in the introduction of modRNA into certain target cells. First, the modRNA must be protected from ribonucleases.[5] This can be accomplished, for example, by wrapping it in liposomes. Such "packaging" can also help to ensure that the modRNA is absorbed into the target cells. This is useful, for example, when used in vaccines, as nanoparticles are taken up by dendritic cells and macrophages, both of which play an important role in activating the immune system.[13]

Furthermore, it may be desirable that the modRNA applied is introduced into specific body cells. This is the case, for example, if heart muscle cells are to be stimulated to multiply. In this case, the packaged modRNA can be injected directly into an artery such as a coronary artery.[14]

Applications

An important field of application are mRNA vaccines.

Replacing uridine with pseudouridine to evade the innate immune system was pioneered by Karikó and Weissman in 2005.[15][16] They won the 2023 Nobel Prize in Physiology or Medicine as a result of their work.[17]

Another milestone was achieved by demonstrating the life-saving efficacy of nucleoside modified mRNA in a mouse model of a lethal lung disease by the team of Kormann and others in 2011.[18]

N1-methyl-pseudouridine was used in vaccine trials against Zika,[19][20][21] HIV-1,[21] influenza,[21] and Ebola[22] in 2017–2018.[23]:5

The first authorized for use in humans were COVID-19 vaccines to address SARS-CoV-2.[24][25][26][27][28][29][30] Examples of COVID-19 vaccines using modRNA include those developed by the cooperation of BioNTech/Pfizer (BNT162b2), and by Moderna (mRNA-1273).[31][32][33] The zorecimeran vaccine developed by Curevac, however, uses unmodified RNA,[34] instead relying on codon optimization to minimize the presence of uridine. This vaccine is less effective, however.[35][16]

Other possible uses of modRNA include the regeneration of damaged heart muscle tissue,[36][37] an enzyme-replacement tool[38] and cancer therapy.[39][40]

References

  1. ↑ "Synthetic chemically modified mRNA (modRNA): toward a new technology platform for cardiovascular biology and medicine". Cold Spring Harbor Perspectives in Medicine 5 (1): a014035. October 2014. doi:10.1101/cshperspect.a014035. PMID 25301935. 
  2. ↑ From DNA to RNA (4 ed.). Garland Science. 2002. https://www.ncbi.nlm.nih.gov/books/NBK26887/. 
  3. ↑ "Nucleoside Modified mRNA Vaccines for Infectious Diseases". RNA Vaccines. Methods in Molecular Biology. 1499. Clifton, N.J.. 2017. pp. 109–121. doi:10.1007/978-1-4939-6481-9_6. ISBN 978-1-4939-6479-6. 
  4. ↑ The Three Roles of RNA in Protein Synthesis (4th ed.). New York: W. H. Freeman. 2000. pp. Sec 4.4. https://www.ncbi.nlm.nih.gov/books/NBK21603. 
  5. ↑ 5.0 5.1 5.2 "Developing mRNA-vaccine technologies". RNA Biology 9 (11): 1319–1330. November 2012. doi:10.4161/rna.22269. PMID 23064118. 
  6. ↑ "RNA mediated Toll-like receptor stimulation in health and disease". RNA Biology 9 (6): 828–842. June 2012. doi:10.4161/rna.20206. PMID 22617878. 
  7. ↑ "Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA". Immunity 23 (2): 165–175. August 2005. doi:10.1016/j.immuni.2005.06.008. PMID 16111635. 
  8. ↑ "N(1)-methylpseudouridine-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: 337–344. November 2015. doi:10.1016/j.jconrel.2015.08.051. PMID 26342664. 
  9. ↑ 9.0 9.1 "N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density". Nucleic Acids Research 45 (10): 6023–6036. June 2017. doi:10.1093/nar/gkx135. PMID 28334758. 
  10. ↑ "Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code". Nucleic Acids Research 44 (2): 852–862. January 2016. doi:10.1093/nar/gkv1182. PMID 26578598. 
  11. ↑ "Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs". RNA 18 (3): 412–420. March 2012. doi:10.1261/rna.028498.111. PMID 22274954. "In contrast, in most archaea this position is occupied by another hypermodified nucleotide: the isosteric N1-methylated pseudouridine.". 
  12. ↑ "Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3' UTRs Identified by Cellular Library Screening". Molecular Therapy 27 (4): 824–836. April 2019. doi:10.1016/j.ymthe.2018.12.011. PMID 30638957. 
  13. ↑ "Nanoparticle vaccines". Vaccine 32 (3): 327–337. January 2014. doi:10.1016/j.vaccine.2013.11.069. PMID 24295808. 
  14. ↑ "Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine". Molecular Therapy. Methods & Clinical Development 9: 330–346. June 2018. doi:10.1016/j.omtm.2018.04.003. PMID 30038937. 
  15. ↑ "Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA". Immunity 23 (2): 165–175. August 2005. doi:10.1016/j.immuni.2005.06.008. PMID 16111635. 
  16. ↑ 16.0 16.1 "The tangled history of mRNA vaccines". Nature 597 (7876): 318–324. September 2021. doi:10.1038/d41586-021-02483-w. PMID 34522017. Bibcode: 2021Natur.597..318D. 
  17. ↑ "The Nobel Prize in Physiology or Medicine 2023". https://www.nobelprize.org/prizes/medicine/2023/summary/. 
  18. ↑ Kormann, Michael S. D.; Hasenpusch, Günther; Aneja, Manish K.; Nica, Gabriela; Flemmer, Andreas W.; Herber-Jonat, Susanne; Huppmann, Marceline; Mays, Lauren E. et al. (February 2011). "Expression of therapeutic proteins after delivery of chemically modified mRNA in mice". Nature Biotechnology 29 (2): 154–157. doi:10.1038/nbt.1733. ISSN 1546-1696. PMID 21217696. https://pubmed.ncbi.nlm.nih.gov/21217696. 
  19. ↑ "Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination". Nature 543 (7644): 248–251. March 2017. doi:10.1038/nature21428. PMID 28151488. Bibcode: 2017Natur.543..248P. "we designed a potent anti-ZIKV vaccine … containing the modified nucleoside 1-methylpseudouridine (m1Ψ)". 
  20. ↑ "Modified mRNA Vaccines Protect against Zika Virus Infection". Cell 168 (6): 1114–1125.e10. March 2017. doi:10.1016/j.cell.2017.02.017. PMID 28222903. "The mRNA was synthesized … where the UTP was substituted with 1-methylpseudoUTP". 
  21. ↑ 21.0 21.1 21.2 "Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses". The Journal of Experimental Medicine 215 (6): 1571–1588. June 2018. doi:10.1084/jem.20171450. PMID 29739835. "In this study, we characterize the immunogenicity of three vaccines consisting of m1Ψ-modified, FPLC-purified mRNA-LNPs encoding HIV-1 envelope (Env), ZIKV prM-E, and influenza virus hemagglutinin (HA)". 
  22. ↑ "Modified mRNA-Based Vaccines Elicit Robust Immune Responses and Protect Guinea Pigs From Ebola Virus Disease". The Journal of Infectious Diseases 217 (3): 451–455. January 2018. doi:10.1093/infdis/jix592. PMID 29281112. "Two mRNA vaccines were synthesized … where the UTP were substituted with 1-methylpseudo UTP". 
  23. ↑ "The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines". Frontiers in Cell and Developmental Biology 9: 789427. 2021-11-04. doi:10.3389/fcell.2021.789427. PMID 34805188. 
  24. ↑ "Pfizer and BioNTech Celebrate Historic First Authorization in the U.S. of Vaccine to Prevent COVID-19". www.businesswire.com. 12 December 2020. https://www.businesswire.com/news/home/20201211005640/en/. 
  25. ↑ "Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine". The New England Journal of Medicine 383 (27): 2603–2615. December 2020. doi:10.1056/NEJMoa2034577. PMID 33301246. 
  26. ↑ "Hoffnungsträger BNT162b2: Wie funktionieren mRNA-Impfstoffe?" (in de). 2020-11-10. https://www.pharmazeutische-zeitung.de/wie-funktionieren-mrna-impfstoffe-121742/seite/alle/. 
  27. ↑ "COVID-19 and mRNA Vaccines-First Large Test for a New Approach". JAMA 324 (12): 1125–1127. September 2020. doi:10.1001/jama.2020.16866. PMID 32880613. 
  28. ↑ "A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates". bioRxiv. 8 September 2020. doi:10.1101/2020.09.08.280818. https://www.biorxiv.org/content/biorxiv/early/2020/09/08/2020.09.08.280818.full.pdf. 
  29. ↑ "Conditions of Authorisation for Pfizer/BioNTech COVID-19 Vaccine". Medicines & Healthcare Products Regulatory Agency. 8 December 2020. https://www.gov.uk/government/publications/regulatory-approval-of-pfizer-biontech-vaccine-for-covid-19/conditions-of-authorisation-for-pfizerbiontech-covid-19-vaccine. 
  30. ↑ Office of the Commissioner (14 December 2020). "Pfizer-BioNTech COVID-19 Vaccine". US FDA. https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/pfizer-biontech-covid-19-vaccine. 
  31. ↑ "SARS-CoV-2 vaccines in development". Nature 586 (7830): 516–527. October 2020. doi:10.1038/s41586-020-2798-3. PMID 32967006. Bibcode: 2020Natur.586..516K. 
  32. ↑ "Moderna's Pipeline". Moderna. https://www.modernatx.com/pipeline. 
  33. ↑ "COVID-19 vaccines poised for launch, but impact on pandemic unclear". Nature Biotechnology: d41587–020–00022-y. November 2020. doi:10.1038/d41587-020-00022-y. PMID 33239758. 
  34. ↑ "COVID-19". CureVac. https://www.curevac.com/en/covid-19/. 
  35. ↑ "CureVac COVID vaccine let-down spotlights mRNA design challenges". Nature 594 (7864): 483. June 2021. doi:10.1038/d41586-021-01661-0. PMID 34145413. Bibcode: 2021Natur.594..483D. 
  36. ↑ "Modified mRNA as a Therapeutic Tool for the Heart". Cardiovascular Drugs and Therapy 34 (6): 871–880. December 2020. doi:10.1007/s10557-020-07051-4. PMID 32822006. 
  37. ↑ "Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction". Nature Biotechnology 31 (10): 898–907. October 2013. doi:10.1038/nbt.2682. PMID 24013197. 
  38. ↑ Ter Huurne, Menno; Parker, Benjamin L.; Liu, Ning Qing; Qian, Elizabeth Ling; Vivien, Celine; Karavendzas, Kathy; Mills, Richard J.; Saville, Jennifer T. et al. (2023-09-07). "GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals". American Journal of Human Genetics 110 (9): 1600–1605. doi:10.1016/j.ajhg.2023.07.013. ISSN 1537-6605. PMID 37607539. 
  39. ↑ "RNA-Based Vaccines in Cancer Immunotherapy". Journal of Immunology Research 2015: 794528. 2015. doi:10.1155/2015/794528. PMID 26665011. 
  40. ↑ "Co-delivery of nucleoside-modified mRNA and TLR agonists for cancer immunotherapy: Restoring the immunogenicity of immunosilent mRNA". Journal of Controlled Release 266: 287–300. November 2017. doi:10.1016/j.jconrel.2017.09.041. PMID 28987878. 

Further reading



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