Nucleoside-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.{{cite journal | vauthors = Chien KR, Zangi L, Lui KO | title = Synthetic chemically modified mRNA (modRNA): toward a new technology platform for cardiovascular biology and medicine | journal = Cold Spring Harbor Perspectives in Medicine | volume = 5 | issue = 1 | pages = a014035 | date = October 2014 | pmid = 25301935 | pmc = 4292072 | doi = 10.1101/cshperspect.a014035 }} 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

File:Ribosome mRNA translation en.svg (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.{{cite book | vauthors = Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P |title=From DNA to RNA |date=2002 |publisher=Garland Science |edition=4 |url=https://www.ncbi.nlm.nih.gov/books/NBK26887/}} 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.{{cite book | vauthors = Pardi N, Weissman D | title = RNA Vaccines | chapter = Nucleoside Modified mRNA Vaccines for Infectious Diseases | series = Methods in Molecular Biology | location = Clifton, N.J. | volume = 1499 | pages = 109–121 | date = 2017 | pmid = 27987145 | doi = 10.1007/978-1-4939-6481-9_6 | isbn = 978-1-4939-6479-6 }}

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.{{cite book | vauthors = Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J |title=The Three Roles of RNA in Protein Synthesis |date=2000 |publisher=W. H. Freeman |location=New York |pages=Sec 4.4 |edition=4th |url=https://www.ncbi.nlm.nih.gov/books/NBK21603}}

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.{{cite journal | vauthors = Schlake T, Thess A, Fotin-Mleczek M, Kallen KJ | title = Developing mRNA-vaccine technologies | journal = RNA Biology | volume = 9 | issue = 11 | pages = 1319–1330 | date = November 2012 | pmid = 23064118 | pmc = 3597572 | doi = 10.4161/rna.22269 }} 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.{{cite journal | vauthors = Dalpke A, Helm M | title = RNA mediated Toll-like receptor stimulation in health and disease | journal = RNA Biology | volume = 9 | issue = 6 | pages = 828–842 | date = June 2012 | pmid = 22617878 | pmc = 3495747 | doi = 10.4161/rna.20206 }}

The inflammatory nature of exogenous RNA can be masked by modifying the nucleosides in mRNA.{{cite journal | vauthors = Karikó K, Buckstein M, Ni H, Weissman D | title = Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA | journal = Immunity | volume = 23 | issue = 2 | pages = 165–175 | date = August 2005 | pmid = 16111635 | doi = 10.1016/j.immuni.2005.06.008 | doi-access = free }} For example, uridine can be replaced with a similar nucleoside such as pseudouridine (Ψ) or N1-methyl-pseudouridine (m1Ψ),{{cite journal | vauthors = Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T | title = N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice | journal = Journal of Controlled Release | volume = 217 | pages = 337–344 | date = November 2015 | pmid = 26342664 | doi = 10.1016/j.jconrel.2015.08.051 | hdl-access = free | hdl = 1854/LU-6993270 | url = https://biblio.ugent.be/publication/6993270 }} and cytosine can be replaced by 5-methylcytosine. Some of these, such as pseudouridine and 5-methylcytosine, occur naturally in eukaryotes,{{cite journal | vauthors = Hoernes TP, Clementi N, Faserl K, Glasner H, Breuker K, Lindner H, Hüttenhofer A, Erlacher MD | display-authors = 6 | title = Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code | journal = Nucleic Acids Research | volume = 44 | issue = 2 | pages = 852–862 | date = January 2016 | pmid = 26578598 | pmc = 4737146 | doi = 10.1093/nar/gkv1182 }} while m1Ψ occurs naturally in archaea.{{cite journal | vauthors = Wurm JP, Griese M, Bahr U, Held M, Heckel A, Karas M, Soppa J, Wöhnert J | display-authors = 6 | title = Identification of the enzyme responsible for N1-methylation of pseudouridine 54 in archaeal tRNAs | journal = RNA | volume = 18 | issue = 3 | pages = 412–420 | date = March 2012 | pmid = 22274954 | doi = 10.1261/rna.028498.111 | pmc = 3285930 | quote = In contrast, in most archaea this position is occupied by another hypermodified nucleotide: the isosteric N1-methylated pseudouridine. }} 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.{{cite journal | vauthors = Svitkin YV, Cheng YM, Chakraborty T, Presnyak V, John M, Sonenberg N | title = N1-methyl-pseudouridine in mRNA enhances translation through eIF2α-dependent and independent mechanisms by increasing ribosome density | journal = Nucleic Acids Research | volume = 45 | issue = 10 | pages = 6023–6036 | date = June 2017 | pmid = 28334758 | pmc = 5449617 | doi = 10.1093/nar/gkx135 }}

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.{{cite journal | vauthors = Orlandini von Niessen AG, Poleganov MA, Rechner C, Plaschke A, Kranz LM, Fesser S, Diken M, Löwer M, Vallazza B, Beissert T, Bukur V, Kuhn AN, Türeci Ö, Sahin U | display-authors = 6 | title = Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3' UTRs Identified by Cellular Library Screening | journal = Molecular Therapy | volume = 27 | issue = 4 | pages = 824–836 | date = April 2019 | pmid = 30638957 | pmc = 6453560 | doi = 10.1016/j.ymthe.2018.12.011 }}

Delivery

File:Comparing uptake of RNA and modRNA by the cell.jpg

Various difficulties are involved in the introduction of modRNA into certain target cells. First, the modRNA must be protected from ribonucleases. 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.{{cite journal | vauthors = Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu C, Middelberg AP | title = Nanoparticle vaccines | journal = Vaccine | volume = 32 | issue = 3 | pages = 327–337 | date = January 2014 | pmid = 24295808 | doi = 10.1016/j.vaccine.2013.11.069 | doi-access = free }}

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.{{cite journal | vauthors = Carlsson L, Clarke JC, Yen C, Gregoire F, Albery T, Billger M, Egnell AC, Gan LM, Jennbacken K, Johansson E, Linhardt G, Martinsson S, Sadiq MW, Witman N, Wang QD, Chen CH, Wang YP, Lin S, Ticho B, Hsieh PC, Chien KR, Fritsche-Danielson R | display-authors = 6 | title = Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection 1 Week Post-myocardial Infarction in Swine | journal = Molecular Therapy. Methods & Clinical Development | volume = 9 | pages = 330–346 | date = June 2018 | pmid = 30038937 | pmc = 6054703 | doi = 10.1016/j.omtm.2018.04.003 }}

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.{{cite journal | vauthors = Karikó K, Buckstein M, Ni H, Weissman D | title = Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA | journal = Immunity | volume = 23 | issue = 2 | pages = 165–175 | date = August 2005 | pmid = 16111635 | doi = 10.1016/j.immuni.2005.06.008 | doi-access = free }}{{cite journal | vauthors = Dolgin E | title = The tangled history of mRNA vaccines | journal = Nature | volume = 597 | issue = 7876 | pages = 318–324 | date = September 2021 | pmid = 34522017 | doi = 10.1038/d41586-021-02483-w | bibcode = 2021Natur.597..318D | s2cid = 237515383 }} They won the 2023 Nobel Prize in Physiology or Medicine as a result of their work.{{cite web | url=https://www.nobelprize.org/prizes/medicine/2023/summary/ | title=The Nobel Prize in Physiology or Medicine 2023 }}

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.{{Cite journal |last1=Kormann |first1=Michael S. D. |last2=Hasenpusch |first2=Günther |last3=Aneja |first3=Manish K. |last4=Nica |first4=Gabriela |last5=Flemmer |first5=Andreas W. |last6=Herber-Jonat |first6=Susanne |last7=Huppmann |first7=Marceline |last8=Mays |first8=Lauren E. |last9=Illenyi |first9=Marta |last10=Schams |first10=Andrea |last11=Griese |first11=Matthias |last12=Bittmann |first12=Iris |last13=Handgretinger |first13=Rupert |last14=Hartl |first14=Dominik |last15=Rosenecker |first15=Joseph |date=February 2011 |title=Expression of therapeutic proteins after delivery of chemically modified mRNA in mice |url=https://pubmed.ncbi.nlm.nih.gov/21217696 |journal=Nature Biotechnology |volume=29 |issue=2 |pages=154–157 |doi=10.1038/nbt.1733 |issn=1546-1696 |pmid=21217696|s2cid=205275040 }}

N1-methyl-pseudouridine was used in vaccine trials against Zika,{{cite journal | vauthors = Pardi N, Hogan MJ, Pelc RS, Muramatsu H, Andersen H, DeMaso CR, Dowd KA, Sutherland LL, Scearce RM, Parks R, Wagner W, Granados A, Greenhouse J, Walker M, Willis E, Yu JS, McGee CE, Sempowski GD, Mui BL, Tam YK, Huang YJ, Vanlandingham D, Holmes VM, Balachandran H, Sahu S, Lifton M, Higgs S, Hensley SE, Madden TD, Hope MJ, Karikó K, Santra S, Graham BS, Lewis MG, Pierson TC, Haynes BF, Weissman D | display-authors = 6 | title = Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination | journal = Nature | volume = 543 | issue = 7644 | pages = 248–251 | date = March 2017 | pmid = 28151488 | pmc = 5344708 | doi = 10.1038/nature21428 | bibcode = 2017Natur.543..248P | quote = we designed a potent anti-ZIKV vaccine … containing the modified nucleoside 1-methylpseudouridine (m1Ψ) }}{{cite journal | vauthors = Richner JM, Himansu S, Dowd KA, Butler SL, Salazar V, Fox JM, Julander JG, Tang WW, Shresta S, Pierson TC, Ciaramella G, Diamond MS | display-authors = 6 | title = Modified mRNA Vaccines Protect against Zika Virus Infection | journal = Cell | volume = 168 | issue = 6 | pages = 1114–1125.e10 | date = March 2017 | pmid = 28222903 | pmc = 5388441 | doi = 10.1016/j.cell.2017.02.017 | quote = The mRNA was synthesized … where the UTP was substituted with 1-methylpseudoUTP }}{{cite journal | vauthors = Pardi N, Hogan MJ, Naradikian MS, Parkhouse K, Cain DW, Jones L, Moody MA, Verkerke HP, Myles A, Willis E, LaBranche CC, Montefiori DC, Lobby JL, Saunders KO, Liao HX, Korber BT, Sutherland LL, Scearce RM, Hraber PT, Tombácz I, Muramatsu H, Ni H, Balikov DA, Li C, Mui BL, Tam YK, Krammer F, Karikó K, Polacino P, Eisenlohr LC, Madden TD, Hope MJ, Lewis MG, Lee KK, Hu SL, Hensley SE, Cancro MP, Haynes BF, Weissman D | display-authors = 6 | title = Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses | journal = The Journal of Experimental Medicine | volume = 215 | issue = 6 | pages = 1571–1588 | date = June 2018 | pmid = 29739835 | pmc = 5987916 | doi = 10.1084/jem.20171450 | quote = 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) }} HIV-1, influenza, and Ebola{{cite journal | vauthors = Meyer M, Huang E, Yuzhakov O, Ramanathan P, Ciaramella G, Bukreyev A | title = Modified mRNA-Based Vaccines Elicit Robust Immune Responses and Protect Guinea Pigs From Ebola Virus Disease | journal = The Journal of Infectious Diseases | volume = 217 | issue = 3 | pages = 451–455 | date = January 2018 | pmid = 29281112 | pmc = 5853918 | doi = 10.1093/infdis/jix592 | quote = Two mRNA vaccines were synthesized … where the UTP were substituted with 1-methylpseudo UTP }} in 2017–2018.{{cite journal | vauthors = Morais P, Adachi H, Yu YT | title = The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines | journal = Frontiers in Cell and Developmental Biology | volume = 9 | pages = 789427 | date = 2021-11-04 | pmid = 34805188 | pmc = 8600071 | doi = 10.3389/fcell.2021.789427 | doi-access = free }}{{Rp|5|quote=further success was obtained with N1-methyl-Ψ-modified mRNA vaccines against HIV-1, Zika, and influenza virus … Ebola virus}}

The first authorized for use in humans were COVID-19 vaccines to address SARS-CoV-2.{{cite news |title=Pfizer and BioNTech Celebrate Historic First Authorization in the U.S. of Vaccine to Prevent COVID-19 |url=https://www.businesswire.com/news/home/20201211005640/en/ |work=www.businesswire.com |date=12 December 2020 }}{{cite journal | vauthors = Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, Perez JL, Pérez Marc G, Moreira ED, Zerbini C, Bailey R, Swanson KA, Roychoudhury S, Koury K, Li P, Kalina WV, Cooper D, Frenck RW, Hammitt LL, Türeci Ö, Nell H, Schaefer A, Ünal S, Tresnan DB, Mather S, Dormitzer PR, Şahin U, Jansen KU, Gruber WC | display-authors = 6 | title = Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine | journal = The New England Journal of Medicine | volume = 383 | issue = 27 | pages = 2603–2615 | date = December 2020 | pmid = 33301246 | pmc = 7745181 | doi = 10.1056/NEJMoa2034577 | s2cid = 228087117 }}{{cite web | vauthors = Hohmann-Jeddi C |title=Hoffnungsträger BNT162b2: Wie funktionieren mRNA-Impfstoffe?|periodical=Pharmazeutische Zeitung|url=https://www.pharmazeutische-zeitung.de/wie-funktionieren-mrna-impfstoffe-121742/seite/alle/|access-date=2020-11-28|date=2020-11-10|language=de}}{{cite journal | vauthors = Abbasi J | title = COVID-19 and mRNA Vaccines-First Large Test for a New Approach | journal = JAMA | volume = 324 | issue = 12 | pages = 1125–1127 | date = September 2020 | pmid = 32880613 | doi = 10.1001/jama.2020.16866 | s2cid = 221476409 | doi-access = free }}{{cite journal | vauthors = Vogel A, Kanevsky I, Che Y, Swanson K, Muik A, Vormehr M, etal |title=A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates |date=8 September 2020 |doi=10.1101/2020.09.08.280818 | journal = bioRxiv |s2cid=221589144 |url=https://www.biorxiv.org/content/biorxiv/early/2020/09/08/2020.09.08.280818.full.pdf }}{{cite web |publisher=Medicines & Healthcare Products Regulatory Agency |url=https://www.gov.uk/government/publications/regulatory-approval-of-pfizer-biontech-vaccine-for-covid-19/conditions-of-authorisation-for-pfizerbiontech-covid-19-vaccine |type=Decision |date=8 December 2020 |title=Conditions of Authorisation for Pfizer/BioNTech COVID-19 Vaccine}}{{cite web | author = Office of the Commissioner |title=Pfizer-BioNTech COVID-19 Vaccine |url=https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/pfizer-biontech-covid-19-vaccine |archive-url=https://web.archive.org/web/20201212030749/https://www.fda.gov/emergency-preparedness-and-response/coronavirus-disease-2019-covid-19/pfizer-biontech-covid-19-vaccine |url-status=dead |archive-date=December 12, 2020 |website=www.fda.gov |type=Decision |publisher=US FDA |date=14 December 2020}} Examples of COVID-19 vaccines using modRNA include those developed by the cooperation of BioNTech/Pfizer (BNT162b2), and by Moderna (mRNA-1273).{{cite journal | vauthors = Krammer F | title = SARS-CoV-2 vaccines in development | journal = Nature | volume = 586 | issue = 7830 | pages = 516–527 | date = October 2020 | pmid = 32967006 | doi = 10.1038/s41586-020-2798-3 | s2cid = 221887746 | doi-access = free | bibcode = 2020Natur.586..516K }}{{cite web|title=Moderna's Pipeline|publisher=Moderna|url=https://www.modernatx.com/pipeline|access-date=2020-11-28}}{{cite journal | vauthors = Dolgin E | title = COVID-19 vaccines poised for launch, but impact on pandemic unclear | journal = Nature Biotechnology | pages = d41587–020–00022-y | date = November 2020 | pmid = 33239758 | doi = 10.1038/d41587-020-00022-y | s2cid = 227176634 }} The zorecimeran vaccine developed by Curevac, however, uses unmodified mRNA,{{Cite web|title=COVID-19|url=https://www.curevac.com/en/covid-19/|access-date=2020-12-21|publisher=CureVac}} instead relying on codon optimization to minimize the presence of uridine. This vaccine is less effective, however.{{cite journal | vauthors = Dolgin E | title = CureVac COVID vaccine let-down spotlights mRNA design challenges | journal = Nature | volume = 594 | issue = 7864 | pages = 483 | date = June 2021 | pmid = 34145413 | doi = 10.1038/d41586-021-01661-0 | bibcode = 2021Natur.594..483D | s2cid = 235480198 | doi-access = free }}

Other possible uses of modRNA include the regeneration of damaged heart muscle tissue,{{cite journal | vauthors = Kaur K, Zangi L | title = Modified mRNA as a Therapeutic Tool for the Heart | journal = Cardiovascular Drugs and Therapy | volume = 34 | issue = 6 | pages = 871–880 | date = December 2020 | pmid = 32822006 | pmc = 7441140 | doi = 10.1007/s10557-020-07051-4 }}{{cite journal | vauthors = Zangi L, Lui KO, von Gise A, Ma Q, Ebina W, Ptaszek LM, Später D, Xu H, Tabebordbar M, Gorbatov R, Sena B, Nahrendorf M, Briscoe DM, Li RA, Wagers AJ, Rossi DJ, Pu WT, Chien KR | display-authors = 6 | title = Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction | journal = Nature Biotechnology | volume = 31 | issue = 10 | pages = 898–907 | date = October 2013 | pmid = 24013197 | pmc = 4058317 | doi = 10.1038/nbt.2682 }} an enzyme-replacement tool{{Cite journal |last1=Ter Huurne |first1=Menno |last2=Parker |first2=Benjamin L. |last3=Liu |first3=Ning Qing |last4=Qian |first4=Elizabeth Ling |last5=Vivien |first5=Celine |last6=Karavendzas |first6=Kathy |last7=Mills |first7=Richard J. |last8=Saville |first8=Jennifer T. |last9=Abu-Bonsrah |first9=Dad |last10=Wise |first10=Andrea F. |last11=Hudson |first11=James E. |last12=Talbot |first12=Andrew S. |last13=Finn |first13=Patrick F. |last14=Martini |first14=Paolo G. V. |last15=Fuller |first15=Maria |date=2023-09-07 |title=GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals |journal=American Journal of Human Genetics |volume=110 |issue=9 |pages=1600–1605 |doi=10.1016/j.ajhg.2023.07.013 |issn=1537-6605 |pmid=37607539|pmc=10502840 }} and cancer therapy.{{cite journal | vauthors = McNamara MA, Nair SK, Holl EK | title = RNA-Based Vaccines in Cancer Immunotherapy | journal = Journal of Immunology Research | volume = 2015 | pages = 794528 | date = 2015 | pmid = 26665011 | pmc = 4668311 | doi = 10.1155/2015/794528 | doi-access = free }}{{cite journal | vauthors = Verbeke R, Lentacker I, Wayteck L, Breckpot K, Van Bockstal M, Descamps B, Vanhove C, De Smedt SC, Dewitte H | display-authors = 6 | title = Co-delivery of nucleoside-modified mRNA and TLR agonists for cancer immunotherapy: Restoring the immunogenicity of immunosilent mRNA | journal = Journal of Controlled Release | volume = 266 | pages = 287–300 | date = November 2017 | pmid = 28987878 | doi = 10.1016/j.jconrel.2017.09.041 | s2cid = 20794075 }}