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Cited by 1RNA and protein synthesis mechanismsSARS-CoV-2 and COVID-19 ResearchRNA Interference and Gene Delivery

A Luciferase-Based Approach for Functional Screening of 5′ and 3′ Untranslated Regions of the mRNA Component for mRNA Vaccines

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Rubtsova, Maria, Mokrushina, Yuliana, Andreev, Dmitry, Poteshnova, Maria, Shepelev, Nikita, Koryagina, Mariya, Moiseeva, Ekaterina, Malabuiok, Diana +8 more·2025·Vaccines
DOI 10.3390/vaccines13050530PMC PMC12115628
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Abstract

Background/Objectives: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation in vivo. The composition of 5′ and 3′ UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for in vivo selection of translationally active UTRs. Methods: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. Results: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. Conclusions: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines.

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  • Inclusion of specific untranslated regions is a promising strategy to enhance stability and efficacy of mRNA therapeutics.

    “Recent studies demonstrate that, in addition to the incorporation of modified nucleosides, the inclusion of specific untranslated regions represents a highly promising strategy to enhance the stability and efficacy of mRNA therapeutics [12][13][14][15][16].”

    From Identification of novel 5’ UTR variants for enhanced mRNA translation and vaccine immunogenicity (2026) · cites this paper as [15]

  • UTRs that provide the most efficient translation initiation, including those derived from highly expressed endogenous genes such as HBA1, may prove suboptimal for inclusion in mRNA vaccine constructs.

    “Therefore, UTRs that provide the most efficient translation initiationincluding those derived from highly expressed endogenous genes, such as HBA1 -may prove suboptimal for inclusion in mRNA vaccine constructs [15].”

    From Identification of novel 5’ UTR variants for enhanced mRNA translation and vaccine immunogenicity (2026) · cites this paper as [15]

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Authors
Rubtsova, M., Mokrushina, Y., Andreev, D., Poteshnova, M., Shepelev, N., Koryagina, M., Moiseeva, E., Malabuiok, D., Prokopenko, Y., Terekhov, S., Chernov, A., Vodovozova, E., Smirnov, I., Dontsova, O., Gabibov, A., & Rubtsov, Y.
Affiliations
  • Russian Academy of Sciences (RU)
  • Lomonosov Moscow State University (RU)
  • Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia
  • Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, Russia
Corresponding author
  • Gabibov, Alexander
  • Rubtsov, Yury
ORCID
Venue
Vaccines
Publisher
MDPI AG
Published
2025-05-16
Volume
13
Issue
5
Pages
530
ISSN
2076-393X (ISSN-L)
DOI
10.3390/vaccines13050530
PMC
PMC12115628
OpenAlex
W4410446307
Cited by
0
References
2
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Vaccines (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Ministry of Science and Higher Education of Russian Federation (#075-15-2021-104)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
mRNA vaccines, 5' untranslated region, 3' untranslated region, nanoluciferase reporter, SARS-CoV-2 spike protein, lipid nanoparticle delivery, translation efficiency, neutralizing antibodies, IgG antibody titers, ribosome profiling

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