Maximizing the Translational Yield of mRNA Therapeutics by Minimizing 5′-UTRs
Abstract
The 5'-untranslated region (5'-UTR) of mRNA contains structural elements, which are recognized by cell-specific RNA-binding proteins, thereby affecting the translation of the molecule. The activation of an innate immune response upon transfection of mRNA into cells is reduced when the mRNA comprises chemically modified nucleotides, putatively by altering the secondary structure of the molecule. Such alteration in the 5'-UTR in turn may affect the functionality of mRNA. In this study, we report on the impact of seven synthetic minimalistic 5'-UTR sequences on the translation of luciferase-encoding unmodified and different chemically modified mRNAs upon transfection in cell culture and in vivo. One minimalistic 5'-UTR, consisting of 14 nucleotides combining the T7 promoter with a Kozak consensus sequence, yielded similar or even higher expression than a 37 nucleotides human alpha-globin 5'-UTR containing mRNA in HepG2 and A549 cells. Furthermore, also the kind of modified nucleotides used in in vitro transcription, affected mRNA translation when using different translation regulators (Kozak vs. translation initiator of short UTRs). The in vitro data were confirmed by bioluminescence imaging of expression in mouse livers, 6 h postintravenous injection of a lipidoid nanoparticle-formulated RNA in female Balb/c mice. Luciferase measurements from liver and spleen showed that minimal 5'-UTRs (3 and 7) were either equally effective or better than human alpha-globin 5'-UTR. These find…
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Specific UTRs, such as one or two copies of the 3′ UTR of globin or a short 14-nucleotide 5′ UTR, increase the translational efficiency of mRNAs.
“numerous works have developed specific UTRs to increase the translational efficiency of mRNAs, as the use of one or two copies of the 3′ UTR of the globin or other known stable mRNA8,9 or short 14-nucleotide 5′ UTR.27”
From Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA (2021) · cites this paper as [27]
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- Authors
- Trepotec, Z., Aneja, M. K., Geiger, J., Hasenpusch, G., Plank, C., & Rudolph, C.
- Affiliations
- Department of Pediatrics, Ludwig-Maximilian-University of Munich, Munich, Germany.
- Ethris (Germany) (DE)
- TUM Klinikum (DE)
- Corresponding author
- Rudolph, Carsten
- Venue
- Tissue Engineering Part A
- Publisher
- SAGE Publications
- Published
- 2019-01
- Volume
- 25
- Issue
- 1-2
- Pages
- 69-79
- ISSN
- 1937-3341 (ISSN-L)
- 1937-335X
- OpenAlex
- W2901057672
- Cited by
- 77
- References
- 10
- Citations by year
- 2026: 9 · 2025: 18 · 2024: 9 · 2023: 12 · 2022: 10 · 2021: 10 · 2020: 4 · 2019: 3 · 2018: 2
- FWCI
- 1.66
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Tissue Engineering Part A (publishedVersion)
- PubMed (publishedVersion)
- mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Keywords
- 5' untranslated region, mRNA translation, chemically modified nucleotides, Kozak sequence, T7 promoter, RNA secondary structure, RNA-binding proteins, innate immune response, lipid nanoparticle delivery, mRNA therapeutics, erythropoietin, luciferase expression, in vivo bioluminescence imaging
- MeSH terms
- A549 Cells, Animals, Female, Humans, Luciferases, Mice, Inbred BALB C, Nucleic Acid Conformation, Protein Biosynthesis, 5' Untranslated Regions, Hep G2 Cells
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