Selection of utrs in MRNA-based gene therapy and vaccines

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Abstract

The untranslated regions (UTRs) of messenger RNAs (mRNAs) play a crucial role in regulating translational efficiency, stability, and tissue-specific expression. The review describes various applications and challenges of UTR design in the development of gene therapy and mRNA-based therapeutics. UTRs affect critical biological functions, such as mRNA stability, modulation of protein synthesis, and attenuation of immune response. Incorporating tissue-specific microRNA (miRNA)-binding sites into 3′ UTRs might improve precise targeting of transgene expression and minimize off-target effects. Nucleotide modifications (pseudouridine, N1-methyladenosine, and N4-acetylcytidine) in mRNA and UTRs in particular, improve mRNA stability and translational efficiency. At the same time, several challenges remain, such as lack of consensus on UTRs best suited for certain biomedical applications. Current efforts are focused on integrating high-throughput screening, computational modeling, and experimental validation to refine UTR-based therapeutic strategies. The review presents current information on the design of UTRs and their role in therapeutic applications, with special focus on the possibilities and limitations of existing approaches.

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About the authors

I. A. Volkhin

Lomonosov Moscow State University

Email: devyatkin_aa@academpharm.ru

Department of Bioengineering and Bioinformatics

Russian Federation, 119234 Moscow

A. Iu. Paremskaia

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies; Pirogov Medical University

Email: devyatkin_aa@academpharm.ru

Department of Biomedicine

Russian Federation, 125315 Moscow; 117997 Moscow

M. A. Dashian

Pirogov Medical University

Email: devyatkin_aa@academpharm.ru
Russian Federation, 117997 Moscow

D. S. Smeshnova

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical

Email: devyatkin_aa@academpharm.ru
Russian Federation, Technologies, 125315 Moscow

R. E. Pavlov

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical

Email: devyatkin_aa@academpharm.ru
Russian Federation, Technologies, 125315 Moscow

O. N. Mityaeva

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies; Moscow Center for Advanced Studies

Email: devyatkin_aa@academpharm.ru
Russian Federation, 125315 Moscow; 123592 Moscow

P. Yu. Volchkov

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies; The MCSC Named After A.S. Loginov

Email: devyatkin_aa@academpharm.ru

Center for Personalized Medicine

Russian Federation, 125315 Moscow; 111123 Moscow

A. A. Deviatkin

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies; Izmerov Research Institute of Occupational Health; Federal State Budgetary Institution ‘Centre for Strategic Planning and Management of Biomedical Health Risks’ of the Federal Medical Biological Agency

Author for correspondence.
Email: devyatkin_aa@academpharm.ru

Laboratory of Postgenomic Technologies

Russian Federation, 125315 Moscow; 105275 Moscow; 119121 Moscow

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2. Fig. 1. Structure of mRNA-based therapeutics: mRNA vaccine (left) and AAV vector-based drug (right). mRNA vaccine contains translation-ready mRNA encapsulated in liposomes. After release from the liposome in the cell, mRNA is translated by the cell's ribosomes. The synthesized protein functions as an antigen stimulating the body's immune response or as a therapeutic protein compensating for defective cellular proteins. AAV vectors are single-stranded DNA encoding a transgene surrounded by inverted terminal repeats (ITRs), a transcription promoter, and a polyadenylation signal (pA). The DNA is enclosed in a viral capsid membrane. After cellular uptake of the vector and removal of the capsid membrane, the AAV gene is transcribed in the nucleus. The resulting mRNA is then exported into the cytoplasm, where translation leads to the synthesis of the therapeutic protein, which carries out its function.

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3. Fig. 2. Schematic representation of regulatory regions in the 5′ UTR and 3′ UTR that affect translation efficiency and mRNA stability. Legend: uORF, upstream open reading frame; lncRNA, long non-coding RNA; RBP, RNA binding protein; miRBS, miRNA binding site; miRISC, miRNA-induced silencing complex.

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4. Fig. 3. Principles of tissue-specific 3′ UTR design. It should be noted that this approach has been successfully implemented using AAV as an example, but its principles can also be applied to mRNA therapy, which opens up prospects for future research. a – The AAV vector expression cassette contains tissue-specific miRNA binding sites in the 3′ UTR. Inclusion of miRBSs for miRNAs present in non-target tissues or cells allows for a decrease in off-target transgene expression. b – The AAV vector enters the cell and enters the nucleus, where the transgene mRNA is transcribed. Following transcription and nuclear export, the tissue-specific miRNA within the miRISC binds to the miRBS in the synthesized mRNA, causing suppression of its expression due to translational repression and/or degradation of the transgene mRNA in non-target cells.

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