A new combination of 5’- and 3’-untranslated regions increases the expression of mRNAs in vitro and in vivo

mRNA vaccine technologies have been actively developing since the beginning of the 21st century and have received a major boost from new findings about the functioning of the immune system and the development of efficient vehicles for nucleic acid delivery. The mRNA vaccine demonstrates superior properties compared to the DNA vaccine, primarily due to accelerated mRNA vaccine development, enhanced flexibility, and the absence of integration into the genome. Artificial mRNAs have biotechnological and medical applications, including the development of antiviral and anticancer mRNA therapeutics. The effective expression of therapeutic mRNA depends upon the appropriate selection of structural elements. Along with the addition of the 5’-cap, appropriate polyadenylation, and sequence codon optimization, 5’- and 3’-untranslated regions (UTRs) play an important role in the translation efficiency of therapeutic mRNAs. In this study, new plasmids containing a novel combination of UTR pairs, namely 5’-UTR-4 and 3’-UTR AES-mtRNR1, were constructed to obtain artificial mRNAs encoding green fluorescent protein (GFP) and firefly luciferase (FLuc) with new structural elements and properties. The novel combination of the UTRs, which is described in this article for the first time, in addition to sufficient polyadenylation and pseudouridinilation of mRNA, was demonstrated to strongly increase the translation of codon-optimized sequences of reporter mRNAs. We generated lipoplexes containing the aforementioned reporter mRNAs and liposomes composed of cationic lipid 2X3 (1,26-bis(cholest-5-en-3beta-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride) and helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). For in vivo experiments, the liposomes were decorated with 2 % of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀). The translation efficiency of mRNAs was found to be superior for the novel UTR combination compared with HBB gene UTRs, both in vitro and in vivo. When mRNA is administered intramuscularly, the proposed combination of UTRs provides lasting expression for more than 4 days. The results demonstrated that the novel UTR pair combination could be useful in the development of artificial mRNAs with enhanced translation efficiency, potentially reducing the dose for mRNA-based therapeutics.

1. Andreev D.E., Dmitriev S.E., Terenin I.M., Prassolov V.S., Merrick W.C., Shatsky I.N. Differential contribution of the m7G-cap to the 5ʹ end-dependent translation initiation of mammalian mRNAs. Nucleic Acids Res. 2009;37(18):6135-6147. doi 10.1093/nar/gkp665

2. Chatterjee S., Pal J.K. Role of 5ʹ- and 3ʹ-untranslated regions of mRNAs in human diseases. Biol Cell. 2009;101(5):251-262. doi 10.1042/BC20080104

3. Chen F., Cocaign-Bousquet M., Girbal L., Nouaille S. 5ʹUTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability. Front Microbiol. 2022;13:1088941. doi 10.3389/fmicb.2022.1088941

4. Conrad T., Plumbom I., Alcobendas M., Vidal R., Sauer S. Maximizing transcription of nucleic acids with efficient T7 promoters. Commun Biol. 2020;3(1):439. doi 10.1038/s42003-020-01167-x

5. Fedorovskiy A.G., Antropov D.N., Dome A.S., Puchkov P.A., Makarova D.M., Konopleva M.V., Matveeva A.M., Panova E.V., Shmendel E.V., Maslov M.A., Dmitriev S.E., Stepanov G.A., Markov O.V. Novel efficient lipid-based delivery systems enable a delayed uptake and sustained expression of mRNA in human cells and mouse tissues. Pharmaceutics. 2024;16(5):684. doi 10.3390/pharmaceutics16050684

6. Gladkikh D.V., Sen’kova A.V., Chernikov I.V., Kabilova T.O., Popova N.A., Nikolin V.P., Shmendel E.V., Maslov M.A., Vlassov V.V., Zenkova M.A., Chernolovskaya E.L. Folate-equipped cationic liposomes deliver anti-MDR1-siRNA to the tumor and increase the efficiency of chemotherapy. Pharmaceutics. 2021;13(8):1252. doi 10.3390/pharmaceutics13081252

7. Hassett K.J., Rajlic I.L., Bahl K., White R., Cowens K., Jacquinet E., Burke K.E. mRNA vaccine trafficking and resulting protein expression after intramuscular administration. Mol Ther Nucleic Acids. 2024;35(1):102083. doi 10.1016/j.omtn.2023.102083

8. Kirshina A.S., Vasileva O.O., Kunyk D.A., Seregina K.K., Muslimov A.R., Ivanov R.A., Reshetnikov V.V. Effects of combinations of untranslated-region sequences on translation of mRNA. Biomolecules. 2023;13(11):1677. doi 10.3390/biom13111677

9. Kozak M. The scanning model for translation: an update. J Cell Biol. 1989;108(2):229-241. doi 10.1083/jcb.108.2.229

10. Leppek K., Byeon G.W., Kladwang W., Wayment-Steele H.K., Kerr C.H., Xu A.F., Kim D.S., … Participants E., Dormitzer P.R., Solorzano A., Barna M., Das R. Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. Nat Commun. 2022;13(1):1536. doi 10.1038/s41467-022-28776-w

11. Markov O.V., Mironova N.L., Shmendel E.V., Serikov R.N., Morozova N.G., Maslov M.A., Vlassov V.V., Zenkova M.A. Multicomponent mannose-containing liposomes efficiently deliver RNA in murine immature dendritic cells and provide productive anti-tumour response in murine melanoma model. J Control Release. 2015;213: 45-56. doi 10.1016/j.jconrel.2015.06.028

12. Morais P., Adachi H., Yu Y.T. The critical contribution of pseudouridine to mRNA COVID-19 vaccines. Front Cell Dev Biol. 2021;9: 789427. doi 10.3389/fcell.2021.789427

13. Mrksich K., Padilla M.S., Joseph R.A., Han E.L., Kim D., Palanki R., Xu J., Mitchell M.J. Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length. J Biomed Mater Res A. 2024;112(9):1494-1505. doi 10.1002/jbm.a.37705

14. Orlandini von Niessen A.G., Poleganov M.A., Rechner C., Plaschke A., Kranz M.L., Fesser M., Diken M., Lower M., Vallazza B., Beissert T., Bukur V., Kuhn A.N., Tureci O., Sahin U. Improving mRNAbased therapeutic gene delivery by expression-augmenting 3ʹ UTRs identified by cellular library screening. Mol Ther. 2019;27(4):824-836. doi 10.1016/j.ymthe.2018.12.011

15. Panova E.A., Kleymenov D.A., Shcheblyakov D.V., Bykonia E.N., Mazunina E.P., Dzharullaeva A.S., Zolotar A.N., … Dmitriev S.E., Gushchin V.A., Naroditsky B.S., Logunov D.Y., Gintsburg A.L. Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A. Front Immunol. 2023;14:1098302. doi 10.3389/fimmu.2023.1098302

16. Ruiz de los Mozos I., Vergara-Irigaray M., Segura V., Villanueva M., Bitarte N., Saramago M., Domingues S., Arraiano C.M., Fechter P., Romby P., Valle J., Solano C., Lasa I., Toledo-Arana A. Base pairing interaction between 5ʹ- and 3ʹ-UTRs controls icaR mRNA translation in Staphylococcus aureus. PLoS Genet. 2013;9(12):e1004001. doi 10.1371/journal.pgen.1004001

17. Sample P.J., Wang B., Reid D.W., Presnyak V., McFadyen I.J., Morris D.R., Seelig J. Human 5′ UTR design and variant effect prediction from a massively parallel translation assay. Nat Biotechnol. 2019;37(7):803-809. doi 10.1038/s41587-019-0164-5

18. Troy T., Jekic-McMullen D., Sambucetti L., Rice B. Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models. Mol Imaging. 2004;3(1):9-23. doi 10.1162/15353500200403196

19. Vysochinskaya V., Shishlyannikov S., Zabrodskaya Y., Shmendel E., Klotchenko S., Dobrovolskaya O., Gavrilova N., Makarova D., Plotnikova M., Elpaeva E., Gorshkov A., Moshkoff D., Maslov M., Vasin A. Influence of lipid composition of cationic liposomes 2X3- DOPE on mRNA delivery into eukaryotic cells. Pharmaceutics. 2022;15(1):8. doi 10.3390/pharmaceutics15010008

20. Yuzhakova D., Kiseleva E., Shirmanova M., Shcheslavskiy V., Sachkova D., Snopova L., Bederina E., Lukina M., Dudenkova V., Yusu­ balieva G., Belovezhets T., Matvienko D., Baklaushev V. Highly invasive fluorescent/bioluminescent patient-derived orthotopic model of glioblastoma in mice. Front Oncol. 2022;12:897839. doi 10.3389/fonc.2022.897839

21. Zhuang X., Qi Y., Wang M., Yu N., Nan F., Zhang H., Tian M., Li C., Lu H., Jin N. mRNA vaccines encoding the HA protein of influenza A H1N1 virus delivered by cationic lipid nanoparticles induce protective immune responses in mice. Vaccines (Basel). 2020;8(1):123. doi 10.3390/vaccines8010123