RU2831165C1 - NUCLEIC ACID CONTAINING REGULATORY ELEMENTS OF RABBIT β-GLOBIN GENE, mtRNR1 AND EMCV - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a nucleic acid intended for transcription of a nucleic acid sequence coding a peptide or protein. Said nucleic acid contains a sequence which codes 5'-untranslated region of the transcript with SEQ ID NO: 1, and a sequence which codes 3'-untranslated region of the transcript with SEQ ID NO: 2. Present invention also relates to RNA molecules intended for nucleic acid sequence translation.

EFFECT: present invention provides a statistically significant increase in the amount of protein translated from the nucleic acid, containing SEQ ID NO: 1 in 5'-UTR and SEQ ID NO: 2 in 3'-UTR.

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Description

Field of technology

The group of inventions relates to the field of biotechnology, in particular to recombinant nucleic acids that can be used in the production of mRNA-based drugs.

State of the art

Ribonucleic acid (mRNA)-based drugs, especially mRNA vaccines, have been widely established as a promising treatment strategy in immunotherapy. The exceptional advantages of mRNA vaccines, including their high efficacy, reduced incidence and severity of adverse reactions, and relatively low production costs, explain the large number of preclinical and clinical trials of mRNA vaccine drugs against various infectious diseases and cancers. Recent technological advances have mitigated some of the problems that hinder mRNA vaccine development, such as low efficiency, which exists in both gene translation and in vivo delivery. Vaccine efficacy primarily depends on the stability and structure of the developed mRNA.

There are various approaches to solving the problem of increasing the stability and/or translational activity of mRNA. One such approach is the use of heterologous untranslated regions of RNA that can stabilize mRNA and/or increase translation efficiency, which should ultimately lead to an increase in the amount of synthesized protein.

Untranslated regions (UTRs) are non-coding parts of the mRNA sequence located upstream (5' UTR) and downstream (3' UTR) of the mRNA coding region. UTRs are known to be associated with mRNA replication and translation processes, and by interacting with RNA-binding proteins, they can affect mRNA resistance to degradation factors and translation efficiency [Schlake T, Thess A, Fotin-Mleczek M, et al. Developing mRNA-vaccine technologies. RNA Biol. 2012 Nov; 9(11):1319-30. doi: 10.4161/rna.22269; Linares-Fernandez S, Lacroix C, Exposito JY, et al. Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response. Trends Mol Med. 2020 Mar; 26(3):311-323. doi: 10.1016/j.molmed.2019.10.002; Xu S, Yang K, Li R, Zhang L. mRNA Vaccine Era-Mechanisms, Drag Platform and Clinical Prospection. Int J Mol Sci. 2020 Sep 9; 21(18):6582. doi: 10.3390/ijms21186582; Kim SC, Sekhon SS, Shin WR, et al. Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency. Mol Cell Toxicol. 2022; 18(1):l-8. doi:10.1007/s13273-021-00171-4].

Some regulatory elements can enhance protein expression at the translation initiation and posttranscriptional levels [Kaufman RJ. Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors. Proc Natl Acad Sci USA. 1985 Feb; 82(3):689-93. doi: 10.1073/pnas.82.3.689. PMID: 2983309; PMCID: РМС397111].

Among the best known non-translated sequences that lead to high translational efficiency, we can highlight the sequences of globin genes. In particular, human α- and β-globin sequences are used to increase the translational efficiency of heterologous mRNAs [Babendure JR, Babendure JL, Ding JH, et al. Control of mammalian trans1ation by mRNA structure near caps. RNA. 2006 May; 12(5):851-61. doi: 10.1261/rna.2309906; Kozak M. Features in the 5' non-coding sequences of rabbit alpha and beta-globin mRNAs that affect trans1ational efficiency. JMolBiol. 1994 Jan 7; 235(1):95-110. doi: 10.1016/S0022-2836(05)80019-1] and rabbit β-globin [Annweiler A, Hipskind RA, Wirth T. A strategy for efficient in vitro trans1ation of cDNAs using the rabbit beta-globin leader sequence. Nucleic Acids Res. 1991 Jul 11; 19(13):3750. doi: 10.1093/паг/19.13.3750]. It is believed that the 5'-HTO of rabbit β-globin provides efficient cap-dependent translation and does not contain any secondary structures or sequence elements that could negatively affect translation [Kozak M. Features in the 5' non-coding sequences of rabbit alpha and beta-globin mRNAs that affect trans1ational efficiency. J Mol Biol. 1994 Jan 7; 235(1):95-110. doi: 10.1016/S0022-2836(05)80019-1]. Regulatory sequences of human globin genes are also used as 3'-UTR and provide high stability and translational activity of mRNA [Leppek K, Byeon GW, Kladwang W, et al. Combinatorial optimization of mRNA structure, stability, and trans1ation for RNA-based therapeutics. Nat Commun. 2022 Mar 22; 13(1): 1536. doi: 10.1038/s41467-022-28776-w].

In addition to the regulatory elements of human globin genes, the 5'-UTR sequences of heat shock protein 70 (HSP70) and histone H3 are also used as heterologous UTRs [Rubtsova MP, Sizova DV, Dmitriev SE, et al. Distinctive properties of the 5'-untrans1ated region of human hsp70 mRNA. J Biol Chem. 2003 Jun 20; 278(25):22350-6. doi: 10.1074/jbc.M303213200, Morris T, Marashi F, Weber L, et al. Involvement of the 5'-leader sequence in coupling the stability of a human H3 histone mRNA with DNA replication. Proc Natl Acad Sci USA. 1986 Feb;83(4):981-5. doi: 10.1073/pnas.83.4.981]. The ability of the HSP70 5'-HTO to provide a high level of translation of heterologous mRNA is probably associated with the presence of IRES-like elements and optimal GC composition [Schlake T, Thess A, Fotin-Mleczek M, et al. Developing mRNA-vaccine technologies. RNA Biol. 2012 Nov; 9(11):1319-30. doi: 10.4161 /rna.22269, Rubtsova MP, Sizova DV, Dmitriev SE, et al. Distinctive properties of the 5'-untrans1ated region of human hsp70 mRNA. J Biol Chem. 2003 Jun 20; 278(25):22350-6. doi: 10.1074/jbc.M303213200]. The effect of HSP70 5' UTR was observed in several different cell lines [10.1046/j.1432-1327.2001.02064.x], indicating that the effect of 5' UTR is likely not cell type specific. The sequence of the 5' UTR of rabbit β-globin is known [GenBank: V00882.1, positions 224-276].

Summarizing the data on natural 3'-UTRs, it can be concluded that regulatory elements of constitutively (constantly) expressed genes are often used to enhance the translational efficiency of heterologous mRNAs.

mtRNR1 is a mitochondrial noncoding 12S rRNA that is involved in the translation of 13 protein-coding genes in mitochondria [Chinnery PF, Hudson G. Mitochondrial genetics. Br Med Bull. 2013; 106(1):135-59. doi: 10.1093/bmb/ldt017].

AES is a distinct member of the Groucho/Transducin-like split gene enhancer family, regulates androgen receptor transcriptional activity and Notch and Wnt signaling and acts as a tumor suppressor gene [Costa AM, Pereira-Castro I, Ricardo E, et al. GRG5/AES interacts with T-cell factor 4 (TCF4) and downregulates Wnt signaling in human cells and zebrafish embryos. PLoS One. 2013 Jul 1; 8(7):e67694. doi: 10.1371/journal.pone.0067694;].

Orlandini von Niessen et al. showed that 3' UTRs containing mtRNR1 and AES in any order promote increased expression of the upstream gene [RU Patents 2720934 and 2783165; Orlandini von Niessen AG, Poleganov MA, Rechner C, et al. Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3' UTRs Identified by Cellular Library Screening. Mol Ther. 2019 Apr 10; 27(4):824-836. doi: 10.1016/j.ymthe.2018.12.011].

There are few data on the use of the 3' UTR of encephalomyocarditis virus (EMCV), however, the article: Marzbany M and Rasekhian M Effect of encephalomyocarditis virus 3' Untrans1ated region on GFP transient expression: implications in recombinant protein production. Boletin Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, 2020, 19 (6): 542-554 discloses the use of the EMCV 3' UTR to increase the translation efficiency of a heterologous gene. The authors used the 3'-UTR sequence of EMCV, which is a 122-nucleotide sequence taken from position 7713-7835 of the EMCV genome, sequence NC_001479 in the NCBI database https://www.ncbi.nlm.nih.gov, accessed 09/14/2023/ as the 3'-UTR.

Currently, there are two known mRNA vaccines in the world that have reached the stage of commercial use: ModeRNA and BioNTech.

ModeRNA's mRNA vaccines use:

5'-UTR with sequence Seq Id No.:3 [sources: US10881730B2 Seq Id No.:181; Jeong D.-E., McCoy M., Amies K., et al. Assemblies-of-Putative-SARS-CoV2-Spike-Encoding-mRNA-Sequences-for-Vaccines-BNT-162b2-and-mRNA-1273, available online at: https://virological.org/t/assemblies-of-putative-sars-cov2-spike-encoding-mrna-sequences-for-vaccines-bnt-162b2-and-mina-1273/663, accessed 08/21/2023; Xia X. Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and ModeRNA mRNA Vaccines. Vaccines (Basel). 2021 Jul 3; 9(7):734. doi: 10.3390/vaccmes9070734];

3'-UTR with Seq Id No:4 [Jeong D.-E., McCoy M., Artiles K., et al. Assemblies-of-Putative-SARS-CoV2-Spike-Encoding-mRNA-Sequences-for-Vaccines-BNT-162b2-and-mRNA-1273, available online at: https://virological.org/t/assemblies-of-putative-sars-cov2-spike-encoding-mrna-sequences-for-vaccines-bnt-162b2-and-mrna-1273/663, accessed 21.08.2023].

BioNTech's mRNA vaccines use:

5'-UTR with Seq Id No:6 and 3'-UTR with Seq Id No:7 [sources: BioNTech from World Health Organization Messenger RNA Encoding the Full-Length SARS-CoV-2 Spike Glycoprotein. [(accessed on 7 June 2021)];2020 Available online: https://web.archive.org/web/20210105162941/https://mednet-commimities.net/irm/db/media/docs/11889.doc; Jeong D.-E., McCoy M., Artiles K., et al. Assemblies-of-rAiMive-SARS-CoV2-Spike-Encoding-mRNA-Sequences-for-Vaccines-BNT-162b2-and-niRNA-1273, available online at: https://virological.org/t/assemblies-of-putative-sars-cov2-spike-encoding-mma-sequences-for-vaccines-bnt-162b2-and-mrna-1273/663, accessed 21 August 2023; Xia X. Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and ModeRNA mRNA Vaccines. Vaccines (Basel). 2021 Jul 3; 9(7):734. doi: 10.3390/vaccmes9070734]. The 3' UTR consists of the 3' UTR sequence of the AES gene and the 3' UTR sequence of mtRNR1.

In addition to the use of natural regulatory elements, an intensive search is currently underway for an optimal artificial, non-naturally occurring, UTR sequence [Cao J, Novoa EM, Zhang Z, et al. High-throughput 5' UTR engineering for enhanced protein production in non-viral gene therapies. Nat Commun. 2021 Jul 6; 12(1):4138. doi: 10.103 8/s41467-021-24436-7].

Despite the intensive search for regulatory elements for use in heterologous recombinant RNAs, the level of protein translation on the mRNA matrix with the best regulatory elements decreases dramatically within 24 hours after administration of the mRNA preparation [RU Patents No. 2720934 and 2783165]. Therefore, the search for an optimal combination of 5'- and 3'-UTRs that will be more effective than existing analogues is a priority.

The technical problem that the present invention is aimed at solving is to solve at least one of the above-mentioned problems in the prior art.

The essence of the invention

One of the possible technical problems that the present invention may be aimed at solving was the creation of recombinant RNA with increased stability and/or translation efficiency, as well as means for obtaining such RNA.

Insufficient translational efficiency of mRNA molecules leads to a low level of expression of the target protein. Regulatory elements of RNA are responsible for translation efficiency: 5'- and 3'-untranslated regions (UTR). An optimal combination of regulatory elements allows for a significant increase in the expression of the target protein.

The technical result achieved by implementing the present invention consists in a statistically significant increase in the amount of protein translated from a nucleic acid containing Seq Id No. 1 in the 3'-UTR and Seq Id No. 2 in the 3'-UTR, compared to the amount of protein translated from a nucleic acid containing 5'- and 3'-UTR used in the mRNA vaccines of well-known manufacturers: ModeRNA or BioNTech, 72 hours after administration to cells, and compared to the amount of protein translated from a nucleic acid containing 5'- and 3'-UTR ModeRNA 48 hours after administration to mice.

The problem can be solved by creating an RNA molecule containing a combination of untranslated elements that are active in relation to translation efficiency and/or nucleic acid stability.

The problem can be solved by creating a nucleic acid containing, in the direction of transcription 5'→3':

(a) promoter;

(b) a nucleic acid sequence which, when transcribed under the control of promoter (a), encodes the 5' untranslated region of the transcript;

(c) a transcribed nucleic acid sequence encoding a peptide or protein;

(d) a nucleic acid sequence which, when transcribed under the control of promoter (a), encodes the 3' untranslated region of the transcript,

wherein said 3'-untranslated region comprises the sequence Seq Id No:1, and said 3'-untranslated region comprises the sequence Seq Id No:2,

wherein nucleic acid sequences (b)-(d) under the control of promoter (a) can be transcribed to form a common transcript in which the nucleic acid sequences transcribed from nucleic acid sequences (b) and (d) are active in increasing the translation efficiency and/or stability of the nucleic acid sequence transcribed from the transcribed nucleic acid sequence (c).

In one embodiment of the invention, an RNA with increased stability and/or translational efficiency is provided, obtained as a result of transcription using any variant of the nucleic acid molecule described above.

In one embodiment of the invention, the RNA with increased stability comprises in the 5'→3' direction:

(a) 5' untranslated region;

(b) a nucleic acid sequence encoding a peptide or protein;

(c) 3'-untranslated region,

wherein said 5'-untranslated region comprises the sequence Seq Id No:1, and said 3'-untranslated region comprises the sequence Seq Id No:2,

wherein nucleic acid sequences (a) and (b) are active in increasing the translation efficiency and/or stability of a nucleic acid sequence encoding a peptide or protein.

The nucleic acid of the present invention can be used for in vitro synthesis of RNA, which can then be encapsulated in lipid nanoparticles to produce an RNA vaccine.

The invention is illustrated by the following graphic materials:

Fig. 1 shows the results of the assessment of the relative bioluminescence of luciferase synthesized from matrix RNAs with different 5'- and 3'-UTR variants in the DC2.4 cell line.

Data are presented as mean ± standard error of the mean. Statistically significant differences are indicated:

compared with ModeRNA mRNA* - p-values <0.05;

compared to BioNTech mRNA # - p-values <0.05;

Legend: Rabb-mtRNR-EMCV - RNA with 5'-UTR containing Seq Id No.: 1 and 3'-UTR containing Seq Id No.: 2; Moderna - RNA containing Seq Id No.: 3 as 5'-UTR and Seq Id No.: 4 as 3'-UTR; Biontech - RNA containing Seq Id No.: 5 as 5'-UTR and Seq Id No.: 6 as 3'-UTR.

Fig. 2 shows the results of the intravital analysis of absolute bioluminescence of luciferase synthesized from mRNAs with different 5'- and 3'-UTR variants in Balb/c mice. Data are presented as mean ± standard error of the mean. Statistically significant differences compared to mRNA with ModeRNA are indicated by: * - p-values <0.05, ** - p-values <0.01, *** - p-values <0.001.

Legend: Rabb-mtRNR-EMCV - RNA with 5'-UTR containing Seq Id No.: 1 and 3'-UTR containing Seq Id No.: 2; Moderna - RNA containing the sequence Seq Id No.: 3 as 5'-UTR and Seq Id No.: 4 as 3'-UTR.

Detailed description of the invention

Unless otherwise indicated, all terms, designations and other scientific terms used in this application are intended to have meanings commonly understood by those skilled in the art to which the present invention pertains. In some cases, definitions of terms with commonly understood meanings are provided in this application for clarity and/or for quick reference and understanding, and the inclusion of such definitions in this description should not be construed as having a significant difference in the meaning of the term from that commonly understood in the art.

In addition, unless the context otherwise requires, singular terms include plural terms, and plural terms include singular terms. Generally, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and are widely used in the art. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is customary in the art, or as described herein.

The invention is based on an unexpected increase in the translation efficiency on an mRNA matrix containing a combination of a modified 5'-UTR sequence of a rabbit β-globin gene transcript as part of the 5'-untranslated region of the mRNA and a modified sequence of the 3'-untranslated region of the EMCV genome in combination with the 3'-untranslated region of mtRNR1 as part of the 3'-untranslated region.

In one embodiment of the present invention, there is provided a nucleic acid comprising, in the 5'→3' transcriptional direction:

(a) promoter

(b) a nucleic acid sequence which, when transcribed under the control of promoter (a), encodes the 5' untranslated region of the transcript;

(c) a transcribed nucleic acid sequence encoding a peptide or protein;

(d) a nucleic acid sequence which, when transcribed under the control of promoter (a), encodes the 3' untranslated region of the transcript,

wherein said 5'-untranslated region comprises the sequence Seq Id No:1, and said 3'-untranslated region comprises the sequence Seq Id No:2,

wherein nucleic acid sequences (b)-(d) under the control of promoter (a) can be transcribed to form a common transcript in which the nucleic acid sequences transcribed from nucleic acid sequences (b) and (d) are active in increasing the translation efficiency and/or stability of the nucleic acid sequence transcribed from the transcribed nucleic acid sequence (c).

The term "promoter" refers to a nucleic acid sequence that is recognized by RNA polymerase as the starting point for transcription.

The expression "a nucleic acid sequence which, when transcribed, encodes a 5'-untranslated region of a transcript" refers to a nucleic acid sequence of the antisense strand encoding said 5'-untranslated region, which requires that the nucleic acid have a sense strand comprising the same sequence as the 5'-untranslated region of the transcript except for the substitution of uracil for thymine. Similarly, "a nucleic acid sequence which, when transcribed, encodes a 3'-untranslated region of a transcript" refers to a nucleic acid sequence of the antisense strand encoding said 3'-untranslated region, which requires that the nucleic acid have a sense strand comprising the same sequence as the 3'-untranslated region of the transcript except for the substitution of uracil for thymine.

The term "5' untranslated region" or "5' UTR" refers to the leader sequence: the noncoding region of an mRNA located immediately after the promoter but before the coding region. As used herein, the term "5' untranslated region" or "5' UTR" also encompasses the DNA sequence that can be transcribed into an RNA sequence that is the 5' UTR.

The term "3' untranslated region" (3' UTR) refers to the non-coding region of RNA located after the RNA sequence encoding the protein or peptide of interest. As used herein, the term "3' untranslated region" or "3' UTR" also encompasses a DNA sequence that can be transcribed into an RNA sequence that is the 3' UTR.

The term "AES" refers to the amino-terminal enhancer of Split, as well as the AES gene. The protein encoded by this gene belongs to the groucho/TLE protein family, and can function as a homo-oligomer or as a hetero-oligomer with other family members to preferentially repress the expression of genes from other family members.

The term "untranslated region of a gene" refers to the UTR sequence in both the gene itself and its transcript.

The expression "nucleic acid sequence of the 5'-untranslated region of the rabbit β-globin gene" refers to the nucleic acid sequence of 53 nucleotides (sequence from nucleotide 224 to 276 of the V00882 locus of the rabbit β-globin gene according to the NCBI database https://www.ncbi.nlm.nih.gov, accessed 09/13/2023).

The expression "nucleic acid sequence of the 3'-untranslated region of the AES gene" refers to the nucleic acid sequence of 142 nucleotides (sequence from 1218 to 1353 nucleotides of the XM_006722664 locus of the AES gene according to the NCBI database https://www.ncbi.nlm.nih.gov, accessed 08/30/2023).

The term "mtRNR1" refers to mitochondrial 12S RNA and also to the mtRNR1 gene that encodes said RNA.

The expression "mtRNR1 3'-untranslated region nucleic acid sequence" refers to the transcript sequence from nucleotide 760 to 898 of the mitochondrial DNA locus OQ884979 according to the NCBI database https://www.ncbi.nlm.nih, accessed 08/30/2023).

Sequence Seq Id No.: 1 is a modified sequence of the 5'-UTR of the rabbit β-globin gene transcript, consisting of 52 nucleotides and differing from the nucleic acid sequence of the 5'-untranslated region of the rabbit β-globin gene by a deletion of one nucleotide at the 9th position (232nd nucleotide of the V00882 locus of the rabbit β-globin gene according to the NCBI database https://www.ncbi.nlm.nih.gov, accessed 09/13/2023.

The sequence of Seq Id No.: 2 is the sequence of 3'-UTR of mtRNR1 modified by deletion of 75th adenine, connected to the sequence of 3'-UTR of EMCV, which is the sequences of the regulatory element of the EMCV genome from 680 to 845 nucleotides of the NC_001479 locus according to the NCBI database https://www.ncbi.nlm.nih, accessed 09/14/2023.

In one embodiment of the invention, an RNA with increased stability and/or translational efficiency is provided, obtained as a result of transcription using any variant of the nucleic acid molecule described above. The RNA of the present invention can be obtained by any method of transcription from a nucleic acid template known from the prior art. Such methods are known to a person skilled in the art, the choice of a specific one is not essential and depends on the goals and objectives of the study or the personal preferences of the specialist.

In one embodiment of the invention, the RNA with increased stability comprises in the 5'→3' direction:

(a) 5' untranslated region;

(b) a nucleic acid sequence encoding a peptide or protein;

(c) 3'-untranslated region,

wherein said 5'-untranslated region comprises the sequence Seq Id No:1, and said 3'-untranslated region comprises the sequence Seq Id No:2,

wherein nucleic acid sequences (a) and (b) are active in increasing the translation efficiency and/or stability of a nucleic acid sequence encoding a peptide or protein.

The nucleic acid of the present invention can be used for in vitro synthesis of RNA, which can then be encapsulated in lipid nanoparticles to produce an RNA vaccine.

The methods for introducing RNA according to the present invention into living cells, vectors for such introduction, encapsulation in lipid nanoparticles and the composition of lipid nanoparticles themselves, described in the examples of the implementation of the present invention, are not essential, do not affect the technical result of the invention and do not limit the scope of the rights of the present invention.

The use of a nucleic acid according to the present invention, containing a combination of the sequences Seq Id No.: 1 in the 5'-UTR and Seq Id No.: 2 in the 3'-UTR, demonstrates an increase in the amount of translated protein compared to other variants, including commercially available ones, at least 72 hours after the introduction of mRNA into cells and 48 hours after the introduction of mRNA into mice.

The essence and industrial applicability of the invention are explained by the following examples of the invention.

Example 1. Obtaining a 5'-UTR construct containing the sequence Seq Id No.: 1 and a 3'-UTR construct containing the sequence Seq Id No.: 2.

The pSmart plasmid (Lucigen, USA) containing the sequence of the T7 promoter, the ampicillin resistance gene, the gene encoding firefly (Photinus pyralis) luciferase, and a sequence of 110 adenines (poly(A) tail) after the 3'-UTR was used as the insertion vector.

The target sequence was amplified by ligating the DNA fragments using overlapping PCR. The target sequence amplicons encoding 5'-UTR and 3'-UTR were then fused to the sequence encoding firefly (Photinus pyralis) luciferase, which was amplified from the pSmart vector, using overlapping PCR. The resulting amplicon was 5'-UTR-luciferase-3'-UTR. The EcoRI (G/AATTC) and BglII (A/GATCT) restriction site sequences were then added to the resulting amplicon using flanking PCR. The resulting amplicon with introduced sites for DNA restriction endonucleases was incubated with restriction endonucleases EcoRI and BglII, purified on an agarose gel and ligated with a similarly prepared commercial vector pSmart (Lucigen, USA).

The Escherichia coli NEB-stable strain (New England Biolabs, UK) was used for transformation. Clones were selected by PCR from colonies and the insert sequence was confirmed by sequencing. E. coli was cultivated to produce the verified plasmid on a shaker-incubator at 30°C and 180 rpm. Then, plasmid DNA was isolated from bacterial cells using the QIAGEN Plasmid Maxi Kit (Qiagen, USA). The resulting plasmid preparation was linearized by the unique Spel restriction site with subsequent visualization in an agarose gel.

Example 2. In vitro mRNA transcription

From the plasmid template obtained as described in Example 1, mRNA was synthesized in vitro.

In vitro transcription was performed in a buffer containing 20 mM dithiothreitol (DTT), 2 mM spermidine, 80 mM HEPES-KOH pH 7.4, 24 mM MgCl ₂ . The reaction mixture also contained 3 mM of each ribonucleoside triphosphate (Biosan, Novosibirsk), 12 mM ARCA cap analog (Biolabmix, Novosibirsk), as well as the remaining components of the transcription reaction, based on 100 μl of the reaction: 40 units of RiboCare ribonuclease inhibitor (Eurogen, Moscow), 500 units of T7 RNA polymerase (Biolabmix, Novosibirsk), 5 μg of linearized plasmid and 1 μl of the enzyme mixture from the RiboMAX Large Scale RNA Production System kit (Promega, USA) as a source of inorganic pyrophosphatase. The reaction was carried out for 2 hours at 37°C, after which 3 mM of each ribonucleoside triphosphate was added to the reaction and the reaction was further incubated for 2 hours. DNA was hydrolyzed using RQ1 nuclease (Promega, USA), RNA was precipitated by adding LiCl to a concentration of 0.32 M and EDTA pH 8.0 to a concentration of 20 mM, followed by incubation on ice for an hour. Then the solution was centrifuged for 15 minutes at 25,000 g and 4°C. The RNA precipitate was washed with 70% ethanol, dissolved in ultrapure water and reprecipitated with alcohol using the standard method. RNA concentration was determined spectrophotometrically by optical density (OD) at a wavelength of 260 nm.

Example 3. In vitro translation efficiency on DC2.4 antigen-presenting cells

The efficiency of in vitro translation was assessed by the amount of synthesized protein (firefly luciferase, Photinus pyralis) with mRNA in the culture of mouse dendritic cells DC2.4. Protein bioluminescence after addition of the substrate was measured 4, 8, 24 and 72 hours after the introduction of mRNA. The transfection agent lipofectamine (Lipofectamine® 3000, Thermo Fisher Scientific) was used as a vector for delivering mRNA to cell cultures.

DC2.4 cells were grown in RPMI-1640 medium (Capricorn) containing 10% FBS (HyClone), 50 μM beta-mercaptoethanol, alanyl-glutamine and vitamins. One day before transfection, DC2.4 cells were seeded into wells of a 96-well plate containing 0.1 ml of culture medium at a density of 30 thousand cells per well. All mRNAs were diluted to a concentration of 100 ng/μl, aliquoted, frozen and stored at -80°C. For transfection, a solution of reference mRNA and experimental mRNA was prepared in a separate tube in sterile phosphate buffer (pH = 7.5) at a rate of 25 μl of buffer and 50 ng of each RNA per well. The reference mRNA contained the Nanoluc luciferase sequence (Seq Id No.: 8) and was used as an internal control for luciferase expression. Next, a solution of the Lipofectamine transfection reagent (Lipofectamine® 3000, Thermo Fisher Scientific) was prepared in a separate tube in sterile phosphate buffer pH 7.5 at a rate of 0.25 μl of the reagent and 25 μl of PBS per well. According to the manufacturer's protocol, the reagent was incubated in PBS at room temperature for 10 minutes, after which 25 μl of the PBS solution containing the experimental and reference mRNA were mixed with 25 μl of the transfection agent solution, and the mixture was incubated for 15 minutes. Next, the transfection mixture in a volume of 50 μl per well was added dropwise to the cells and the cells were incubated for 4, 8, 24 or 72 hours. Then, the conditioned medium was removed, the cells were washed with a phosphate buffer pH 7.5, removed from the substrate, lysed using the Dual Luciferase Assay lysis buffer (Promega, USA) and the luciferase bioluminescence intensity was analyzed using the Dual Luciferase Assay kit (Promega, USA). For comparison, mRNAs differing in 5'- and 3'-UTR were used: mRNA with UTR from ModeRNA contained the 5'-UTR sequence Seq Id No. 3 and the 3'-UTR sequence Seq Id No. 4, mRNA with UTR from BioNTech contained the 5'-UTR sequence Seq Id No. 6 and the 3'-UTR sequence Seq Id No. 7. mRNAs were obtained as described in Examples 1 and 2. The results are shown in Fig. 1.

In vitro results on the DC2.4 cell line indicate that the bioluminescence intensity after transfection of mRNA with regulatory elements according to the present invention was higher at all time points compared to BioNTech (p<0.05), and also higher compared to mRNA with ModeRNA regulatory elements 72 hours after transfection (p=0.05). The bioluminescence intensity reflects the translation efficiency, thus the obtained results indicate the achievement of the technical result in vitro.

Statistical analysis of the data was performed using one-factor analysis of variance (ANOVA). Pairwise comparisons were performed using Fisher's exact test (Fisher LSD as post hoc). Statistical calculations were performed in STATISTICA 8.

Example 4. Creation of lipid nanoparticles (LNPs) containing mRNA

Encapsulation of mRNA into lipid nanoparticles was performed by mixing an aqueous solution of mRNA in 10 mM citrate buffer (pH 3.0) with an alcoholic solution of the lipid mixture. The lipid mixture contained the following components: ionizable cationic lipid ALC-0315 (BroadPharm, USA), distearoylphosphatidylcholine (DSPC) (Avanti Polar Lipids, USA), cholesterol (Sigma-Aldrich, USA), DMG-PEG-2000 (BroadPharm, USA) in a molar ratio of 46.3:9.4:42.7:1.6. The mass fraction of mRNA in LNPs was 0.04% by weight. Mixing was performed in a microfluidic cartridge on The NanoAssemblr™ Benchtop device (Precision Nanosystems, USA) according to the standard method [Prikazchikova TA, Abakumova TO, Sergeeva OV, et al. Design and Validation of siRNA Targeting Gankyrin in the Murine Liver. Russ J Bioorg Chem., 2021, 47:441-446].

To form particles, the ethanol lipid solution was mixed with the mRNA solution in 10 mM citrate buffer, pH 3.0, at a ratio of 1:3 by volume at a flow rate of 10 ml/min in The NanoAssemblr™ Benchtop microfluidic cartridge. The formed particles were then dialyzed against a 500-fold volume of phosphate-buffered saline, pH 7.4, at 4°C for 12 hours. The particles obtained after dialysis were sterile filtered through a polyethersulfone membrane with a pore diameter of 0.2 μm and stored at 4°C if necessary. After filtration, the quality of the obtained particles was analyzed by two parameters: particle size and mRNA content in the particles. Particle size was determined by dynamic light scattering (DLS) using a ZetasizerNano ZSP instrument (Malvern Panalitycal, USA). The mRNA content in the particles was determined by the difference in the fluorescent signal level when staining the particle suspension with the RiboGreen reagent (Thermo Fischer Scientific, USA) before and after their destruction. The Triton X-100 detergent (Sigma-Aldrich, USA) was used to destroy the particles.

Example 5. Translation efficiency in vivo

The efficiency of in vitro translation was assessed by the amount of synthesized protein (firefly luciferase, Photinus pyralis) with mRNA in Balb/c mice. Lipid nanoparticles obtained as described in Example 4 were used as a vector for mRNA delivery to mice.

The experiment was performed on 8-9 week old male Balb/c mice. The mice were intramuscularly injected with lipid nanoparticles containing 10 μg of mRNA encoding Photinus pyralis firefly luciferase with different 5'- and 3'-UTRs. Bioluminescence was assessed 4, 8, 24, 48 and 72 hours after administration of LNPs with mRNA. Twenty minutes before bioluminescence assessment, the mice were intraperitoneally injected with 15 mg/ml D-luciferin solution (Goldbio, USA) at a rate of 10 μl per 1 g of animal body weight. The animals were then anesthetized with 2.0% Isoflurane (Laboratories Karizoo, S.A., Spain) mixed with oxygen for 5 minutes. Intravital luminescence analysis was performed using an IVIS Lumina Series III instrument (PerkinElmer, USA). The results of the analysis are presented in Fig. 2.

In vivo results in BALB/c mice showed that the bioluminescence intensity after administration of mRNA with regulatory elements of the present invention translating firefly luciferase (FLuc) was higher compared to mRNA with regulatory regions of ModeRNA at 24 hours (t=2.36, p=0.028) after administration.

The obtained results showed that the introduction of mRNA with 5'-UTR containing the sequence Seq Id No.: 1 and 3'-UTR containing the sequence Seq Id No.: 2 to mice leads to a higher efficiency of luciferase synthesis and an increase in the amount of protein compared to mRNA with ModeRNA regulatory elements. After 72 hours, the intensity of bioluminescence after the introduction of the compared mRNAs did not differ statistically, which can be interpreted as the fact that the expression result after the introduction of mRNA with regulatory elements according to the present invention is at least not lower than mRNA with ModeRNA regulatory elements. mRNA with regulatory elements from BioNTech was not used for the experiment on a living model, since it showed worse results in the in vitro experiment.

Statistical analysis of the data was performed using the Student's T test. Statistical calculations were performed in the STATISTICA 8 program.

The above examples, without limiting the scope of the rights of the present invention, confirm the achievement of the technical result in the implementation of the present invention, namely, a statistically significant increase in the amount of protein translated from a nucleic acid containing Seq Id No.: 1 in the 5'-UTR and Seq Id No.: 2 in the 3'-UTR, compared to the amount of protein translated from a nucleic acid containing 5'- and 3'-UTR used in the composition of mRNA vaccines from well-known manufacturers: ModeRNA or BioNTech, 72 hours after administration to cells, and compared to the amount of protein translated from a nucleic acid containing 5'- and 3'-UTR ModeRNA 48 hours after administration to mice.

All publications, patents and patent applications are incorporated herein by reference. Although the invention has been described in the foregoing description with respect to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that some details described herein may be substantially modified without departing from the spirit of the invention.

The use of singular terms in the context of the description of the invention shall be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The terms "consisting of," "having," "including," and "comprising" shall be construed as open-ended terms, i.e., meaning "including, but not limited to," unless otherwise indicated. The recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each distinct value falling within that range unless otherwise indicated herein, and each distinct value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or illustrative language (e.g., "such as") provided herein is merely intended to better describe the invention and does not impose a limitation on the scope of the invention unless otherwise stated. No language in the description should be construed as indicating any unreported element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these embodiments may become apparent to those skilled in the art upon reading the preceding description. The inventors expect skilled artisans to employ such variations as the circumstances dictate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the features set forth in the appended claims as permitted by applicable law. Moreover, any combination of the above-described features in all their possible variations is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The applicant requests that the submitted application materials “Nucleic acid containing regulatory elements of the rabbit β-globin gene, mtRNR1 and EMCV” be considered for the purpose of issuing a patent for the invention.

--->

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Claims (19)

1. A nucleic acid intended for transcription of a nucleic acid sequence encoding a peptide or protein, containing in the transcription direction 5’→3’: (a) promoter; (b) a nucleic acid sequence which, when transcribed under the control of promoter (a), encodes the 5' untranslated region of the transcript; (c) a transcribed nucleic acid sequence encoding a peptide or protein; (d) a nucleic acid sequence which, when transcribed under the control of promoter (a), encodes the 3'-untranslated region of the transcript, wherein said 5'-untranslated region comprises the sequence Seq Id No: 1, and said 3'-untranslated region comprises the sequence Seq Id No: 2, wherein nucleic acid sequences (b)-(d) under the control of promoter (a) can be transcribed to form a common transcript in which the nucleic acid sequences transcribed from nucleic acid sequences (b) and (d) are active in increasing the translation efficiency and/or stability of the nucleic acid sequence transcribed from the transcribed nucleic acid sequence (c). 2. RNA with increased stability, intended for translation of a nucleic acid sequence encoding a peptide or protein, obtained as a result of transcription using a nucleic acid molecule according to claim 1 as a template, wherein said RNA contains, in the 5’→3’ direction: (a) 5'-untranslated region; (b) a nucleic acid sequence encoding a peptide or protein; (c) 3'-untranslated region, wherein said 5'-untranslated region comprises the sequence Seq Id: 1, and said 3'-untranslated region comprises the sequence Seq Id: 2, wherein nucleic acid sequences (a) and (b) are active in increasing the translation efficiency and/or stability of a nucleic acid sequence encoding a peptide or protein. 3. RNA intended for translation of a nucleic acid sequence encoding a peptide or protein, containing, in the 5’→3’ direction: (a) 5'-untranslated region; (b) a nucleic acid sequence encoding a peptide or protein; (c) 3'-untranslated region, wherein said 5'-untranslated region comprises the sequence Seq Id No: 1, and said 3'-untranslated region comprises the sequence Seq Id No: 2, wherein nucleic acid sequences (a) and (b) are active in increasing the translation efficiency and/or stability of a nucleic acid sequence encoding a peptide or protein.