mRNA medicine: Recent progresses in chemical modification, design, and engineering

Mascola, J. R.; Fauci, A. S. Novel vaccine technologies for the 21st century. Nat. Rev. Immunol. 2020, 20, 87–88.

Brenner, S.; Jacob, F.; Meselson, M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 1961, 190, 576–581.

Wolff, J. A.; Malone, R. W.; Williams, P.; Chong, W.; Acsadi, G.; Jani, A.; Felgner, P. L. Direct gene transfer into mouse muscle in vivo. Science 1990, 247, 1465–1468.

Pardi, N.; Hogan, M. J.; Porter, F. W.; Weissman, D. mRNA vaccines-a new era in vaccinology. Nat. Rev. Drug Discov. 2018, 17, 261–279.

Polack, F. P.; Thomas, S. J.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Perez, J. L.; Pérez Marc, G.; Moreira, E. D.; Zerbini, C. et al. Safety and efficacy of the BNT162b2 mRNA covid-19 vaccine. N. Engl. J. Med. 2020, 383, 2603–2615.

Baden, L. R.; El Sahly, H. M.; Essink, B.; Kotloff, K.; Frey, S.; Novak, R.; Diemert, D.; Spector, S. A.; Rouphael, N.; Creech, C. B. et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N. Engl. J. Med. 2021, 384, 403–416.

Wilson, E.; Goswami, J.; Baqui, A. H.; Doreski, P. A.; Perez-Marc, G.; Zaman, K.; Monroy, J.; Duncan, C. J. A.; Ujiie, M.; Rämet, M. et al. Efficacy and safety of an mRNA-based RSV PreF vaccine in older adults. N. Engl. J. Med. 2023, 389, 2233–2244.

Sabnis, S.; Kumarasinghe, E. S.; Salerno, T.; Mihai, C.; Ketova, T.; Senn, J. J.; Lynn, A.; Bulychev, A.; McFadyen, I.; Chan, J. et al. A novel amino lipid series for mRNA delivery: Improved endosomal escape and sustained pharmacology and safety in non-human primates. Mol. Ther. 2018, 26, 1509–1519.

Weber, J. S.; Carlino, M. S.; Khattak, A.; Meniawy, T.; Ansstas, G.; Taylor, M. H.; Kim, K. B.; McKean, M.; Long, G. V.; Sullivan, R. J. et al. Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): A randomised, phase 2b study. Lancet 2024, 403, 632–644.

Mackensen, A.; Haanen, J. B. A. G.; Koenecke, C.; Alsdorf, W.; Wagner-Drouet, E.; Borchmann, P.; Heudobler, D.; Ferstl, B.; Klobuch, S.; Bokemeyer, C. et al. CLDN6-specific CAR-T cells plus amplifying RNA vaccine in relapsed or refractory solid tumors: The phase 1 BNT211-01 trial. Nat. Med. 2023, 29, 2844–2853.

Xiao, Y. F.; Tang, Z. M.; Huang, X. G.; Chen, W.; Zhou, J.; Liu, H. J.; Liu, C.; Kong, N.; Tao, W. Emerging mRNA technologies: Delivery strategies and biomedical applications. Chem. Soc. Rev. 2022, 51, 3828–3845.

Xiao, Y. L.; Chen, J.; Zhou, H.; Zeng, X. D.; Ruan, Z. P.; Pu, Z. Y.; Jiang, X. Y.; Matsui, A.; Zhu, L. L.; Amoozgar, Z. et al. Combining p53 mRNA nanotherapy with immune checkpoint blockade reprograms the immune microenvironment for effective cancer therapy. Nat. Commun. 2022, 13, 758.

Yi, J. Z.; Lei, X. L.; Guo, F. T.; Chen, Q. B.; Chen, X. Y.; Zhao, K. T.; Zhu, C. L.; Cheng, X. M.; Lin, J. W.; Yin, H. et al. Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models. Antiviral Res. 2023, 215, 105618.

Karikó, K.; Ni, H. P.; Capodici, J.; Lamphier, M.; Weissman, D. mRNA is an endogenous ligand for toll-like receptor 3. J. Biol. Chem. 2004, 279, 12542–12550.

Heil, F.; Hemmi, H.; Hochrein, H.; Ampenberger, F.; Kirschning, C.; Akira, S.; Lipford, G.; Wagner, H.; Bauer, S. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004, 303, 1526–1529.

Xiong, Q. Q.; Lee, G. Y.; Ding, J. X.; Li, W. L.; Shi, J. J. Biomedical applications of mRNA nanomedicine. Nano Res. 2018, 11, 5281–5309.

Karikó, K.; Muramatsu, H.; Welsh, F. A.; Ludwig, J.; Kato, H.; Akira, S.; Weissman, D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 2008, 16, 1833–1840.

Warminski, M.; Mamot, A.; Depaix, A.; Kowalska, J.; Jemielity, J. Chemical modifications of mRNA ends for therapeutic applications. Acc. Chem. Res. 2023, 56, 2814–2826.

Fu, L. Y.; Zhang, Y.; Farokhzad, R. A.; Mendes, B. B.; Conde, J.; Shi, J. J. 'Passive' nanoparticles for organ-selective systemic delivery: Design, mechanism and perspective. Chem. Soc. Rev. 2023, 52, 7579–7601.

Breda, L.; Papp, T. E.; Triebwasser, M. P.; Yadegari, A.; Fedorky, M. T.; Tanaka, N.; Abdulmalik, O.; Pavani, G.; Wang, Y. P.; Grupp, S. A. et al. In vivo hematopoietic stem cell modification by mRNA delivery. Science 2023, 381, 436–443.

Cheng, Q.; Wei, T.; Farbiak, L.; Johnson, L. T.; Dilliard, S. A.; Siegwart, D. J. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat. Nanotechnol. 2020, 15, 313–320.

Sun, Y. H.; Chatterjee, S.; Lian, X. Z.; Traylor, Z.; Sattiraju, S. R.; Xiao, Y. F.; Dilliard, S. A.; Sung, Y. C.; Kim, M.; Lee, S. M. et al. In vivo editing of lung stem cells for durable gene correction in mice. Science 2024, 384, 1196–1202.

Liu, S.; Cheng, Q.; Wei, T.; Yu, X. L.; Johnson, L. T.; Farbiak, L.; Siegwart, D. J. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing. Nat. Mater. 2021, 20, 701–710.

Lin, Y. X.; Wang, Y.; Ding, J. X.; Jiang, A. P.; Wang, J.; Yu, M.; Blake, S.; Liu, S. S.; Bieberich, C. J.; Farokhzad, O. C. et al. Reactivation of the tumor suppressor PTEN by mRNA nanoparticles enhances antitumor immunity in preclinical models. Sci. Transl. Med. 2021, 13, eaba9772.

Islam, M. A.; Xu, Y. J.; Tao, W.; Ubellacker, J. M.; Lim, M.; Aum, D.; Lee, G. Y.; Zhou, K.; Zope, H.; Yu, M. et al. Restoration of tumour-growth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA. Nat. Biomed. Eng. 2018, 2, 850–864.

Sahin, U.; Karikó, K.; Türeci, Ö. mRNA-based therapeutics-developing a new class of drugs. Nat. Rev. Drug Discov. 2014, 13, 759–780.

Chaudhary, N.; Weissman, D.; Whitehead, K. A. mRNA vaccines for infectious diseases: Principles, delivery and clinical translation. Nat. Rev. Drug Discov. 2021, 20, 817–838.

Hou, X. C.; Zaks, T.; Langer, R.; Dong, Y. Z. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 2021, 6, 1078–1094.

Barbier, A. J.; Jiang, A. Y.; Zhang, P.; Wooster, R.; Anderson, D. G. The clinical progress of mRNA vaccines and immunotherapies. Nat. Biotechnol. 2022, 40, 840–854.

Rohner, E.; Yang, R.; Foo, K. S.; Goedel, A.; Chien, K. R. Unlocking the promise of mRNA therapeutics. Nat. Biotechnol. 2022, 40, 1586–1600.

Conde, J.; Langer, R.; Rueff, J. mRNA therapy at the convergence of genetics and nanomedicine. Nat. Nanotechnol. 2023, 18, 537–540.

Nance, K. D.; Meier, J. L. Modifications in an emergency: The role of N1-methylpseudouridine in COVID-19 vaccines. ACS Cent. Sci. 2021, 7, 748–756.

Mulroney, T. E.; Pöyry, T.; Yam-Puc, J. C.; Rust, M.; Harvey, R. F.; Kalmar, L.; Horner, E.; Booth, L.; Ferreira, A. P.; Stoneley, M. et al. N¹-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Nature 2024, 625, 189–194.

Despic, V.; Jaffrey, S. R. mRNA ageing shapes the Cap2 methylome in mammalian mRNA. Nature 2023, 614, 358–366.

Zhou, F.; Huang, L. J.; Li, S. Q.; Yang, W. F.; Chen, F. M.; Cai, Z. X.; Liu, X. L.; Xu, W. J.; Lehto, V. P.; Lächelt, U. et al. From structural design to delivery: mRNA therapeutics for cancer immunotherapy. Exploration 2024, 4, 20210146.

Zhang, H.; Zhang, L.; Lin, A.; Xu, C. C.; Li, Z. Y.; Liu, K. B.; Liu, B. X.; Ma, X. P.; Zhao, F. F.; Jiang, H. L. et al. Algorithm for optimized mRNA design improves stability and immunogenicity. Nature 2023, 621, 396–403.

Santer, L.; Bär, C.; Thum, T. Circular RNAs: A novel class of functional RNA molecules with a therapeutic perspective. Mol. Ther. 2019, 27, 1350–1363.

Kim, D. Y.; Atasheva, S.; McAuley, A. J.; Plante, J. A.; Frolova, E. I.; Beasley, D. W. C.; Frolov, I. Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs. Proc. Natl. Acad. Sci. USA 2014, 111, 10708–10713.

Beissert, T.; Perkovic, M.; Vogel, A.; Erbar, S.; Walzer, K. C.; Hempel, T.; Brill, S.; Haefner, E.; Becker, R.; Türeci, Ö. et al. A trans-amplifying RNA vaccine strategy for induction of potent protective immunity. Mol. Ther. 2020, 28, 119–128.

Usman, N.; Cedergren, R. Exploiting the chemical synthesis of RNA. Trends Biochem. Sci. 1992, 17, 334–339.

Rong, M. Q.; He, B.; McAllister, W. T.; Durbin, R. K. Promoter specificity determinants of T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 1998, 95, 515–519.

Obi, P.; Chen, Y. G. The design and synthesis of circular RNAs. Methods 2021, 196, 85–103.

Magadum, A.; Kaur, K.; Zangi, L. mRNA-based protein replacement therapy for the heart. Mol. Ther. 2019, 27, 785–793

E, C.; Dai, L. Q.; Yu, J. Switching promotor recognition of phage RNA polymerase in silico along lab-directed evolution path. Biophys. J. 2022, 121, 582–595.

Lu, X. L.; Wu, H.; Xia, H.; Huang, F. T.; Yan, Y.; Yu, B. B.; Cheng, R.; Drulis-Kawa, Z.; Zhu, B. Klebsiella phage KP34 RNA polymerase and its use in RNA synthesis. Front. Microbiol. 2019, 10, 2487

Huang, X. G.; Kong, N.; Zhang, X. C.; Cao, Y. H.; Langer, R.; Tao, W. The landscape of mRNA nanomedicine. Nat. Med. 2022, 28, 2273–2287.

To, K. K. W.; Cho, W. C. S. An overview of rational design of mRNA-based therapeutics and vaccines. Expert Opin. Drug Discov. 2021, 16, 1307–1317.

Qin, S. G.; Tang, X. S.; Chen, Y. T.; Chen, K. P.; Fan, N.; Xiao, W.; Zheng, Q.; Li, G. H.; Teng, Y. Q.; Wu, M. et al. mRNA-based therapeutics: Powerful and versatile tools to combat diseases. Signal Transduct. Target. Ther. 2022, 7, 166.

Van Hoecke, L.; Roose, K. How mRNA therapeutics are entering the monoclonal antibody field. J. Transl. Med. 2019, 17, 54.

Wadhwa, A.; Aljabbari, A.; Lokras, A.; Foged, C.; Thakur, A. Opportunities and challenges in the delivery of mRNA-based vaccines. Pharmaceutics 2020, 12, 102.

de Mey, W.; De Schrijver, P.; Autaers, D.; Pfitzer, L.; Fant, B.; Locy, H.; Esprit, A.; Lybaert, L.; Bogaert, C.; Verdonck, M. et al. A synthetic DNA template for fast manufacturing of versatile single epitope mRNA. Mol. Ther. Nucl. Acids 2022, 29, 943–954.

Kis, Z.; Shattock, R.; Shah, N.; Kontoravdi, C. Emerging technologies for low-cost, rapid vaccine manufacture. Biotechnol. J. 2019, 14, 1–2.

Chow, K. T.; Gale, M. Jr.; Loo, Y. M. RIG-I and other RNA sensors in antiviral immunity. Annu. Rev. Immunol. 2018, 36, 667–694.

Loo, Y. M.; Gale, M. Jr. Immune signaling by RIG-I-like receptors. Immunity 2011, 34, 680–692.

Hornung, V.; Ellegast, J.; Kim, S.; Brzózka, K.; Jung, A.; Kato, H.; Poeck, H.; Akira, S.; Conzelmann, K. K.; Schlee, M. et al. 5'-Triphosphate RNA is the ligand for RIG-I. Science 2006, 314, 994–997.

Buggert, M.; Höglund, P. The prize of prizes: mRNA research paving the way for COVID-19 vaccine success wins the Nobel Prize in Physiology or Medicine 2023. Scand. J. Immunol. 2023, 98, e13340.

Diebold, S. S.; Massacrier, C.; Akira, S.; Paturel, C.; Morel, Y.; Sousa, C. R. E. Nucleic acid agonists for toll-like receptor 7 are defined by the presence of uridine ribonucleotides. Eur. J. Immunol. 2006, 36, 3256–3267.

Karikó, K.; Buckstein, M.; Ni, H. P.; Weissman, D. Suppression of RNA recognition by toll-like receptors: The impact of nucleoside modification and the evolutionary origin of RNA. Immunity 2005, 23, 165–175.

Anderson, B. R.; Muramatsu, H.; Nallagatla, S. R.; Bevilacqua, P. C.; Sansing, L. H.; Weissman, D.; Karikó, K. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 2010, 38, 5884–5892.

Andries, O.; Cafferty, S. M.; De Smedt, S. C.; Weiss, R.; Sanders, N. N.; Kitada, T. N¹-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J. Control. Release 2015, 217, 337–344.

Weng, Y. H.; Li, C. H.; Yang, T. R.; Hu, B.; Zhang, M. J.; Guo, S.; Xiao, H. H.; Liang, X. J.; Huang, Y. Y. The challenge and prospect of mRNA therapeutics landscape. Biotechnol. Adv. 2020, 40, 107534.

Kauffman, K. J.; Mir, F. F.; Jhunjhunwala, S.; Kaczmarek, J. C.; Hurtado, J. E.; Yang, J. H.; Webber, M. J.; Kowalski, P. S.; Heartlein, M. W.; DeRosa, F. et al. Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo. Biomaterials 2016, 109, 78–87.

Sittplangkoon, C.; Alameh, M. G.; Weissman, D.; Lin, P. J. C.; Tam, Y. K.; Prompetchara, E.; Palaga, T. mRNA vaccine with unmodified uridine induces robust type I interferon-dependent anti-tumor immunity in a melanoma model. Front. Immunol. 2022, 13, 983000.

Shanmugasundaram, M.; Senthilvelan, A.; Kore, A. R. Recent advances in modified cap analogs: Synthesis, biochemical properties, and mRNA based vaccines. Chem. Rec. 2022, 22, e202200005.

Ramanathan, A.; Robb, G. B.; Chan, S. H. mRNA capping: Biological functions and applications. Nucleic Acids Res. 2016, 44, 7511–7526.

Ziemniak, M.; Strenkowska, M.; Kowalska, J.; Jemielity, J. Potential therapeutic applications of RNA cap analogs. Future Med. Chem. 2013, 5, 1141–1172.

Sonenberg, N.; Gingras, A. C. The mRNA 5' cap-binding protein eIF4E and control of cell growth. Curr. Opin. Cell Biol. 1998, 10, 268–275.

Grudzien, E.; Stepinski, J.; Jankowska-Anyszka, M.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E. Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency. RNA 2004, 10, 1479–1487.

Li, Y.; Kiledjian, M. Regulation of mRNA decapping. Wiley Interdiscip. Rev. RNA 2010, 1, 253–265.

Devarkar, S. C.; Wang, C.; Miller, M. T.; Ramanathan, A.; Jiang, F. G.; Khan, A. G.; Patel, S. S.; Marcotrigiano, J. Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I. Proc. Natl. Acad. Sci. USA 2016, 113, 596–601.

Schlake, T.; Thess, A.; Thran, M.; Jordan, I. mRNA as novel technology for passive immunotherapy. Cell. Mol. Life Sci. 2019, 76, 301–328.

Sikorski, P. J.; Warminski, M.; Kubacka, D.; Ratajczak, T.; Nowis, D.; Kowalska, J.; Jemielity, J. The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells. Nucleic Acids Res. 2020, 48, 1607–1626.

Muttach, F.; Muthmann, N.; Rentmeister, A. Synthetic mRNA capping. Beilstein J. Org. Chem. 2017, 13, 2819–2832.

Linares-Fernández, S.; Lacroix, C.; Exposito, J. Y.; Verrier, B. Tailoring mRNA vaccine to balance innate/adaptive immune response. Trends Mol. Med. 2020, 26, 311–323.

Fuchs, A. L.; Neu, A.; Sprangers, R. A general method for rapid and cost-efficient large-scale production of 5' capped RNA. RNA 2016, 22, 1454–1466.

Miao, L.; Zhang, Y.; Huang, L. mRNA vaccine for cancer immunotherapy. Mol. Cancer 2021, 20, 41.

Wojtczak, B. A.; Sikorski, P. J.; Fac-Dabrowska, K.; Nowicka, A.; Warminski, M.; Kubacka, D.; Nowak, E.; Nowotny, M.; Kowalska, J.; Jemielity, J. 5'-Phosphorothiolate dinucleotide cap analogues: Reagents for messenger RNA modification and potent small-molecular inhibitors of decapping enzymes. J. Am. Chem. Soc. 2018, 140, 5987–5999.

Blakney, A. K.; Ip, S.; Geall, A. J. An update on self-amplifying mRNA vaccine development. Vaccines 2021, 9, 97.

Peng, Z. H.; Sharma, V.; Singleton, S. F.; Gershon, P. D. Synthesis and application of a chain-terminating dinucleotide mRNA cap analog. Org. Lett. 2002, 4, 161–164.

Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R. E. Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG. RNA 2001, 7, 1486–1495.

Strenkowska, M.; Kowalska, J.; Lukaszewicz, M.; Zuberek, J.; Su, W.; Rhoads, R. E.; Darzynkiewicz, E.; Jemielity, J. Towards mRNA with superior translational activity: Synthesis and properties of ARCA tetraphosphates with single phosphorothioate modifications. New J. Chem. 2010, 34, 993–1007.

Warminski, M.; Trepkowska, E.; Smietanski, M.; Sikorski, P. J.; Baranowski, M. R.; Bednarczyk, M.; Kedzierska, H.; Majewski, B.; Mamot, A.; Papiernik, D. et al. Trinucleotide mRNA cap analogue N6-benzylated at the site of posttranscriptional ^m6A_m mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo. J. Am. Chem. Soc. 2024, 146, 8149–8163.

Kawaguchi, D.; Kodama, A.; Abe, N.; Takebuchi, K.; Hashiya, F.; Tomoike, F.; Nakamoto, K.; Kimura, Y.; Shimizu, Y.; Abe, H. Phosphorothioate modification of mRNA accelerates the rate of translation initiation to provide more efficient protein synthesis. Angew. Chem., Int. Ed. 2020, 59, 17403–17407.

Klöcker, N.; Weissenboeck, F. P.; van Dülmen, M.; Špaček, P.; Hüwel, S.; Rentmeister, A. Photocaged 5' cap analogues for optical control of mRNA translation in cells. Nat. Chem. 2022, 14, 905–913.

Tang, T. T. L.; Passmore, L. A. Recognition of poly(A) RNA through its intrinsic helical structure. Cold Spring Harb. Symp. Quant. Biol. 2019, 84, 21–30.

Körner, C. G.; Wahle, E. Poly(A) tail shortening by a mammalian poly(A)-specific 3'-exoribonuclease. J. Biol. Chem. 1997, 272, 10448–10456.

Goss, D. J.; Kleiman, F. E. Poly(A) binding proteins: Are they all created equal. Wiley Interdiscip. Rev. RNA 2013, 4, 167–179.

Hershey, J. W. B. Regulation of protein synthesis and the role of eIF3 in cancer. Braz. J. Med. Biol. Res. 2010, 43, 920–930.

Pelletier, J.; Sonenberg, N. The organizing principles of eukaryotic ribosome recruitment. Annu. Rev. Biochem. 2019, 88, 307–335.

Meijer, H. A.; Bushell, M.; Hill, K.; Gant, T. W.; Willis, A. E.; Jones, P.; de Moor, C. H. A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells. Nucleic Acids Res. 2007, 35, e132.

Viegas, I. J.; de Macedo, J. P.; Serra, L.; De Niz, M.; Temporão, A.; Pereira, S. S.; Mirza, A. H.; Bergstrom, E.; Rodrigues, J. A.; Aresta-Branco, F. et al. N⁶-methyladenosine in poly(A) tails stabilize VSG transcripts. Nature 2022, 604, 362–370.

Hinnebusch, A. G.; Ivanov, I. P.; Sonenberg, N. Translational control by 5'-untranslated regions of eukaryotic mRNAs. Science 2016, 352, 1413–1416.

Babendure, J. R.; Babendure, J. L.; Ding, J. H.; Tsien, R. Y. Control of mammalian translation by mRNA structure near caps. RNA 2006, 12, 851–861.

Gómez-Aguado, I.; Rodríguez-Castejón, J.; Vicente-Pascual, M.; Rodríguez-Gascón, A.; Solinís, M. Á.; del Pozo-rodríguez, A. Nanomedicines to deliver mRNA: State of the art and future perspectives. Nanomaterials 2020, 10, 364.

Matoulkova, E.; Michalova, E.; Vojtesek, B.; Hrstka, R. The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells. RNA Biol. 2012, 9, 563–576.

Kozak, M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 1987, 196, 947–950.

Adibzadeh, S.; Fardaei, M.; Takhshid, M. A.; Miri, M. R.; Rafiei Dehbidi, G.; Farhadi, A.; Ranjbaran, R.; Alavi, P.; Nikouyan, N.; Seyyedi, N. et al. Enhancing stability of destabilized green fluorescent protein using chimeric mRNA containing human beta-globin 5' and 3' untranslated regions. Avicenna J. Med. Biotechnol. 2019, 11, 112–117.

Fotin-Mleczek, M.; Duchardt, K. M.; Lorenz, C.; Pfeiffer, R.; Ojkić-Zrna, S.; Probst, J.; Kallen, K. J. Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity. J. Immunother. 2011, 34, 1–15.

Truong, B.; Allegri, G.; Liu, X. B.; Burke, K. E.; Zhu, X. L.; Cederbaum, S. D.; Häberle, J.; Martini, P. G. V.; Lipshutz, G. S. Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency. Proc. Natl. Acad. Sci. USA 2019, 116, 21150–21159.

Gustafsson, C.; Govindarajan, S.; Minshull, J. Codon bias and heterologous protein expression. Trends Biotechnol. 2004, 22, 346–353.

Novoa, E. M.; de Pouplana, L. R. Speeding with control: Codon usage, tRNAs, and ribosomes. Trends Genet. 2012, 28, 574–581.

Cannarozzi, G.; Schraudolph, N. N.; Faty, M.; von Rohr, P.; Friberg, M. T.; Roth, A. C.; Gonnet, P.; Gonnet, G.; Barral, Y. A role for codon order in translation dynamics. Cell 2010, 141, 355–367.

Presnyak, V.; Alhusaini, N.; Chen, Y. H.; Martin, S.; Morris, N.; Kline, N.; Olson, S.; Weinberg, D.; Baker, K. E.; Graveley, B. R. et al. Codon optimality is a major determinant of mRNA stability. Cell 2015, 160, 1111–1124.

Vaidyanathan, S.; Azizian, K. T.; Haque, A. K. M.; Henderson, J. M.; Hendel, A.; Shore, S.; Antony, J. S.; Hogrefe, R. I.; Kormann, M. S. D.; Porteus, M. H. et al. Uridine depletion and chemical modification increase cas9 mRNA activity and reduce immunogenicity without HPLC purification. Mol. Ther. Nucl. Acids 2018, 12, 530–542.

Zhang, J. J.; Liu, Y. H.; Li, C.; Xiao, Q.; Zhang, D. D.; Chen, Y.; Rosenecker, J.; Ding, X. Y.; Guan, S. Recent advances and innovations in the preparation and purification of in vitro-transcribed-mRNA-based molecules. Pharmaceutics 2023, 15, 2182.

Mauro, V. P.; Chappell, S. A. A critical analysis of codon optimization in human therapeutics. Trends Mol. Med. 2014, 20, 604–613.

Weissman, D. mRNA transcript therapy. Expert Rev. Vaccines 2015, 14, 265–281.

Spencer, P. S.; Siller, E.; Anderson, J. F.; Barral, J. M. Silent substitutions predictably alter translation elongation rates and protein folding efficiencies. J. Mol. Biol. 2012, 422, 328–335.

Mauger, D. M.; Cabral, B. J.; Presnyak, V.; Su, S. V.; Reid, D. W.; Goodman, B.; Link, K.; Khatwani, N.; Reynders, J.; Moore, M. J. et al. mRNA structure regulates protein expression through changes in functional half-life. Proc. Natl. Acad. Sci. USA 2019, 116, 24075–24083.

Kudla, G.; Lipinski, L.; Caffin, F.; Helwak, A.; Zylicz, M. High guanine and cytosine content increases mRNA levels in mammalian cells. PLoS Biol. 2006, 4, e180.

Konu, Ö.; Li, M. D. Correlations between mRNA expression levels and GC contents of coding and untranslated regions of genes in rodents. J. Mol. Evol. 2002, 54, 35–41.

Sample, P. J.; Wang, B.; Reid, D. W.; Presnyak, V.; McFadyen, I. J.; Morris, D. R.; Seelig, G. Human 5' UTR design and variant effect prediction from a massively parallel translation assay. Nat. Biotechnol. 2019, 37, 803–809.

Cao, J. C.; Novoa, E. M.; Zhang, Z. Z.; Chen, W. C. W.; Liu, D. B.; Choi, G. C. G.; Wong, A. S. L.; Wehrspaun, C.; Kellis, M.; Lu, T. K. High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies. Nat. Commun. 2021, 12, 4138.

Linder, J.; Seelig, G. Fast activation maximization for molecular sequence design. BMC Bioinformatics 2021, 22, 510.

Linder, J.; Bogard, N.; Rosenberg, A. B.; Seelig, G. A generative neural network for maximizing fitness and diversity of synthetic DNA and protein sequences. Cell Syst. 2020, 11, 49–62.e16.

Brito, L. A.; Kommareddy, S.; Maione, D.; Uematsu, Y.; Giovani, C.; Berlanda Scorza, F.; Otten, G. R.; Yu, D.; Mandl, C. W.; Mason, P. W. et al. Self-amplifying mRNA vaccines. Adv. Genet. 2015, 89, 179–233.

Minnaert, A. K.; Vanluchene, H.; Verbeke, R.; Lentacker, I.; De Smedt, S. C.; Raemdonck, K.; Sanders, N. N.; Remaut, K. Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: Getting the message across. Adv. Drug Deliv. Rev. 2021, 176, 113900.

Papukashvili, D.; Rcheulishvili, N.; Liu, C.; Ji, Y.; He, Y. J.; Wang, P. G. Self-amplifying RNA approach for protein replacement therapy. Int. J. Mol. Sci. 2022, 23, 12884.

Geall, A. J.; Mandl, C. W.; Ulmer, J. B. RNA: The new revolution in nucleic acid vaccines. Semin. Immunol 2013, 25, 152–159

Bloom, K.; van den Berg, F.; Arbuthnot, P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021, 28, 117–129.

Blakney, A. K.; McKay, P. F.; Shattock, R. J. Structural components for amplification of positive and negative strand VEEV splitzicons. Front. Mol. Biosci. 2018, 5, 71.

Ljungberg, K.; Liljeström, P. Self-replicating alphavirus RNA vaccines. Expert Rev. Vaccines 2015, 14, 177–194.

Schmidt, C.; Schnierle, B. S. Self-amplifying RNA vaccine candidates: Alternative platforms for mRNA vaccine development. Pathogens 2023, 12, 138.

Maruggi, G.; Zhang, C. L.; Li, J. W.; Ulmer, J. B.; Yu, D. mRNA as a transformative technology for vaccine development to control infectious diseases. Mol. Ther. 2019, 27, 757–772.

Lin, G. B.; Yan, H.; Sun, J.; Zhao, J. C.; Zhang, Y. Self-replicating RNA nanoparticle vaccine elicits protective immune responses against SARS-CoV-2. Mol. Ther. Nucl. Acids 2023, 32, 650–666.

Blakney, A. K.; McKay, P. F.; Bouton, C. R.; Hu, K.; Samnuan, K.; Shattock, R. J. Innate inhibiting proteins enhance expression and immunogenicity of self-amplifying RNA. Mol. Ther. 2021, 29, 1174–1185.

Meo, S. A.; Bukhari, I. A.; Akram, J.; Meo, A. S.; Klonoff, D. C. COVID-19 vaccines: Comparison of biological, pharmacological characteristics and adverse effects of Pfizer/BioNTech and Moderna Vaccines. Eur. Rev. Med. Pharmacol. Sci. 2021, 25, 1663–1669.

Vogel, A. B.; Lambert, L.; Kinnear, E.; Busse, D.; Erbar, S.; Reuter, K. C.; Wicke, L.; Perkovic, M.; Beissert, T.; Haas, H. et al. Self-amplifying RNA vaccines give equivalent protection against influenza to mRNA vaccines but at much lower doses. Mol. Ther. 2018, 26, 446–455.

Dolgin, E. Self-copying RNA vaccine wins first full approval: What's next. Nature 2023, 624, 236–237.

Oda, Y.; Kumagai, Y.; Kanai, M.; Iwama, Y.; Okura, I.; Minamida, T.; Yagi, Y.; Kurosawa, T.; Greener, B.; Zhang, Y. et al. Immunogenicity and safety of a booster dose of a self-amplifying RNA COVID-19 vaccine (ARCT-154) versus BNT162b2 mRNA COVID-19 vaccine: A double-blind, multicentre, randomised, controlled, phase 3, non-inferiority trial. Lancet Infect. Dis. 2024, 24, 351–360.

Geall, A. J.; Verma, A.; Otten, G. R.; Shaw, C. A.; Hekele, A.; Banerjee, K.; Cu, Y.; Beard, C. W.; Brito, L. A.; Krucker, T. et al. Nonviral delivery of self-amplifying RNA vaccines. Proc. Natl. Acad. Sci. USA 2012, 109, 14604–14609.

Spuul, P.; Balistreri, G.; Hellström, K.; Golubtsov, A. V.; Jokitalo, E.; Ahola, T. Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells. J. Virol. 2011, 85, 4739–4751.

Schmidt, C.; Haefner, E.; Gerbeth, J.; Beissert, T.; Sahin, U.; Perkovic, M.; Schnierle, B. S. A taRNA vaccine candidate induces a specific immune response that protects mice against Chikungunya virus infections. Mol. Ther. Nucl. Acids 2022, 28, 743–754.

Schmidt, C.; Hastert, F. D.; Gerbeth, J.; Beissert, T.; Sahin, U.; Perkovic, M.; Schnierle, B. S. A bivalent trans-amplifying RNA vaccine candidate induces potent Chikungunya and ross river virus specific immune responses. Vaccines 2022, 10, 1374.

Hyde, J. L.; Chen, R. B.; Trobaugh, D. W.; Diamond, M. S.; Weaver, S. C.; Klimstra, W. B.; Wilusz, J. The 5' and 3' ends of alphavirus RNAs-Non-coding is not non-functional. Virus Res. 2015, 206, 99–107.

Liu, X.; Zhang, Y.; Zhou, S. R.; Dain, L.; Mei, L.; Zhu, G. Z. Circular RNA: An emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines. J. Control. Release 2022, 348, 84–94.

Nigro, J. M.; Cho, K. R.; Fearon, E. R.; Kern, S. E.; Ruppert, J. M.; Oliner, J. D.; Kinzler, K. W.; Vogelstein, B. Scrambled exons. Cell 1991, 64, 607–613.

Jeck, W. R.; Sharpless, N. E. Detecting and characterizing circular RNAs. Nat. Biotechnol. 2014, 32, 453–461.

Wilusz, J. E. A 360° view of circular RNAs: From biogenesis to functions. Wiley Interdiscip. Rev. RNA 2018, 9, e1478.

Westholm, J. O.; Miura, P.; Olson, S.; Shenker, S.; Joseph, B.; Sanfilippo, P.; Celniker, S. E.; Graveley, B. R.; Lai, E. C. Genome-wide analysis of Drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation. Cell Rep. 2014, 9, 1966–1980.

Chen, L. L. The expanding regulatory mechanisms and cellular functions of circular RNAs. Nat. Rev. Mol. Cell Biol. 2020, 21, 475–490.

Filbin, M. E.; Kieft, J. S. Toward a structural understanding of IRES RNA function. Curr. Opin. Struct. Biol. 2009, 19, 267–276.

Yang, Y.; Fan, X. J.; Mao, M. W.; Song, X. W.; Wu, P.; Zhang, Y.; Jin, Y. F.; Yang, Y.; Chen, L. L.; Wang, Y. et al. Extensive translation of circular RNAs driven by N⁶-methyladenosine. Cell Res. 2017, 27, 626–641.

Qu, L.; Yi, Z. Y.; Shen, Y.; Lin, L. R.; Chen, F.; Xu, Y. Y.; Wu, Z. G.; Tang, H. X.; Zhang, X. X.; Tian, F. et al. Circular RNA vaccines against SARS-CoV-2 and emerging variants. Cell 2022, 185, 1728–1744.

Liu, X.; Abraham, J. M.; Cheng, Y. L.; Wang, Z. X.; Wang, Z.; Zhang, G. J.; Ashktorab, H.; Smoot, D. T.; Cole, R. N.; Boronina, T. N. et al. Synthetic circular RNA functions as a miR-21 sponge to suppress gastric carcinoma cell proliferation. Mol. Ther. Nucl. Acids 2018, 13, 312–321.

Chen, Y. G.; Kim, M. V.; Chen, X. Q.; Batista, P. J.; Aoyama, S.; Wilusz, J. E.; Iwasaki, A.; Chang, H. Y. Sensing self and foreign circular RNAs by intron identity. Mol. Cell 2017, 67, 228–238.e5.

Wang, Z.; Ma, K.; Cheng, Y. L.; Abraham, J. M.; Liu, X.; Ke, X. Q.; Wang, Z. R.; Meltzer, S. J. Synthetic circular multi-miR sponge simultaneously inhibits miR-21 and miR-93 in esophageal carcinoma. Lab. Invest. 2019, 99, 1442–1453.

Schreiner, S.; Didio, A.; Hung, L. H.; Bindereif, A. Design and application of circular RNAs with protein-sponge function. Nucleic Acids Res. 2020, 48, 12326–12335.

Zhang, L. L.; Liang, D. W.; Chen, C. M.; Wang, Y.; Amu, G.; Yang, J. L.; Yu, L. J.; Dmochowski, I. J.; Tang, X. J. Circular siRNAs for reducing off-target effects and enhancing long-term gene silencing in cells and mice. Mol. Ther. Nucl. Acids 2018, 10, 237–244.

Zhang, L. L.; Liang, D. W.; Wang, Y.; Li, D.; Zhang, J. H.; Wu, L.; Feng, M. K.; Yi, F.; Xu, L. Z.; Lei, L. D. et al. Caged circular siRNAs for photomodulation of gene expression in cells and mice. Chem. Sci. 2018, 9, 44–51.

Dolinnaya, N. G.; Sokolova, N. I.; Ashirbekova, D. T.; Shabarova, Z. A. The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide. Nucleic Acids Res. 1991, 19, 3067–3072.

Chen, X. J.; Lu, Y. Circular RNA: Biosynthesis in vitro. Front. Bioeng. Biotechnol. 2021, 9, 787881.

Chen, C. Y.; Sarnow, P. Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 1995, 268, 415–417.

Gholamalipour, Y.; Karunanayake Mudiyanselage, A.; Martin, C. T. 3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses. Nucleic Acids Res. 2018, 46, 9253–9263.

Lang, K.; Micura, R. The preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments. Nat. Protoc. 2008, 3, 1457–1466.

Petkovic, S.; Müller, S. RNA circularization strategies in vivo and in vitro. Nucleic Acids Res. 2015, 43, 2454–2465.

Nandakumar, J.; Ho, C. K.; Lima, C. D.; Shuman, S. RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2. J. Biol. Chem. 2004, 279, 31337–31347.

Chen, H.; Cheng, K.; Liu, X. L.; An, R.; Komiyama, M.; Liang, X. G. Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand. Nucleic Acids Res. 2020, 48, e54.

Puttaraju, M.; Been, M. Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucleic Acids Res. 1992, 20, 5357–5364.

Ford, E.; Ares, M. Jr. Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4. Proc. Natl. Acad. Sci. USA 1994, 91, 3117–3121.

Rausch, J. W.; Heinz, W. F.; Payea, M. J.; Sherpa, C.; Gorospe, M.; Le Grice, S. F. J. Characterizing and circumventing sequence restrictions for synthesis of circular RNA in vitro. Nucleic Acids Res. 2021, 49, e35.

Xu, S. F.; Xu, Y.; Solek, N. C.; Chen, J. G.; Gong, F. L.; Varley, A. J.; Golubovic, A.; Pan, A. N.; Dong, S. T.; Zheng, G. et al. Tumor-tailored ionizable lipid nanoparticles facilitate IL-12 circular RNA delivery for enhanced lung cancer immunotherapy. Adv. Mater. 2024, 36, 2400307.

Li, H. J.; Peng, K.; Yang, K.; Ma, W. B.; Qi, S. L.; Yu, X. Y.; He, J.; Lin, X.; Yu, G. C. Circular RNA cancer vaccines drive immunity in hard-to-treat malignancies. Theranostics 2022, 12, 6422–6436.

Jarrell, K. A. Inverse splicing of a group II intron. Proc. Natl. Acad. Sci. USA 1993, 90, 8624–8627.

Mikheeva, S.; Hakim-Zargar, M.; Carlson, D.; Jarrell, K. Use of an engineered ribozyme to produce a circular human exon. Nucleic Acids Res. 1997, 25, 5085–5094.

Wesselhoeft, R. A.; Kowalski, P. S.; Anderson, D. G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 2018, 9, 2629.

Müller, S.; Appel, B. In vitro circularization of RNA. RNA Biol. 2017, 14, 1018–1027

Zhang, J.; Luo, Z.; Zheng, Y.; Duan, M. Y.; Qiu, Z. J.; Huang, C. CircRNA as an Achilles heel of cancer: Characterization, biomarker and therapeutic modalities. J. Transl. Med. 2024, 22, 752.

Aditham, A.; Shi, H. L.; Guo, J. T.; Zeng, H.; Zhou, Y. M.; Wade, S. D.; Huang, J. H.; Liu, J.; Wang, X. Chemically modified mocRNAs for highly efficient protein expression in mammalian cells. ACS Chem. Biol. 2022, 17, 3352–3366.

Qiu, M.; Tang, Y.; Chen, J. J.; Muriph, R.; Ye, Z. F.; Huang, C. F.; Evans, J.; Henske, E. P.; Xu, Q. B. Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis. Proc. Natl. Acad. Sci. USA 2022, 119, e2116271119.

Zhao, X. W.; Chen, J. J.; Qiu, M.; Li, Y. M.; Glass, Z.; Xu, Q. B. Imidazole-based synthetic lipidoids for in vivo mRNA delivery into primary T lymphocytes. Angew. Chem., Int. Ed. 2020, 59, 20083–20089.

Melamed, J. R.; Yerneni, S. S.; Arral, M. L.; LoPresti, S. T.; Chaudhary, N.; Sehrawat, A.; Muramatsu, H.; Alameh, M. G.; Pardi, N.; Weissman, D. et al. Ionizable lipid nanoparticles deliver mRNA to pancreatic β cells via macrophage-mediated gene transfer. Sci. Adv. 2023, 9, eade1444.

Rurik, J. G.; Tombácz, I.; Yadegari, A.; Méndez Fernández, P.; Shewale, S. V.; Li, L.; Kimura, T.; Soliman, O. Y.; Papp, T. E.; Tam, Y. K. et al. CAR T cells produced in vivo to treat cardiac injury. Science 2022, 375, 91–96.

Green, A. A.; Silver, P. A.; Collins, J. J.; Yin, P. Toehold switches: De-novo-designed regulators of gene expression. Cell 2014, 159, 925–939.

Zhao, E. M.; Mao, A. S.; de Puig, H.; Zhang, K. H.; Tippens, N. D.; Tan, X.; Ran, F. A.; Han, I.; Nguyen, P. Q.; Chory, E. J. et al. RNA-responsive elements for eukaryotic translational control. Nat. Biotechnol. 2022, 40, 539–545.

Plotkin, J. B.; Robins, H.; Levine, A. J. Tissue-specific codon usage and the expression of human genes. Proc. Natl. Acad. Sci. USA 2004, 101, 12588–12591.

Jain, R.; Frederick, J. P.; Huang, E. Y.; Burke, K. E.; Mauger, D. M.; Andrianova, E. A.; Farlow, S. J.; Siddiqui, S.; Pimentel, J.; Cheung-Ong, K. et al. MicroRNAs enable mRNA therapeutics to selectively program cancer cells to self-destruct. Nucl. Acid Ther. 2018, 28, 285–296.