Trinucleotide capping reagent, method for producing same and use

Abstract

The group of inventions relates to the field of molecular biology, virology and medicine, and more particularly to novel reagents for the cotranscriptional capping of synthetic mRNA and to a method for producing same. The proposed reagent can be used to produce functional mRNA containing a 5' cap structure analog. The group of inventions makes it possible to create an mRNA cap structure analog that is cotranscriptionally incorporated into the structure of the mRNA and provides for more efficient mRNA translation.

Description

[0001] Trinucleotide capping reagent, method of its production and application.

[0002] Field of technology

[0003] The invention relates to molecular biology, virology, and medicine, specifically to new reagents for cotranscriptional capping of synthetic mRNA and a method for producing them. The proposed reagent can be used to produce functional mRNA containing an analogue of the 5'-cap structure.

[0004] State of the art

[0005] The technological breakthrough associated with mRNA has opened a new era in drug development. mRNA-based drugs are expected to become the third major class of drugs after small molecules and antibodies in the near future [D. Qiongyu, H. Tianyu, Z. Qiuxia, J. Xueying, C. Feng, Ch. Xiaodong. How far are the new wave of mRNA drugs from us? mRNA product current perspective and future development. Frontiers in Immunology, v. 13, 2022, doi=10.3389 / fimmu.2022.974433, ISSN=1664-3224].

[0006] Currently, two mRNA-based drugs are approved for use: vaccines against COVID-19 produced by Moderna and Phizer / BioNTech. Both vaccines have demonstrated high efficacy and are being used in many countries worldwide. Additionally, vaccines against influenza, AIDS, rabies, Zika, cytomegalovirus infection, metapneumovirus infection, and other infectious diseases are in various stages of clinical trials. Anticancer drugs based on mRNA are being actively developed (against acute myeloid leukemia, colorectal cancer, melanoma, glioblastoma, malignant mesothelioma, for the treatment of liver and brain metastases, etc.).Studies are being conducted on mRNA-based therapeutic drugs for the treatment of genetic diseases (cystic fibrosis, propionic acidemia, methylmalonic acidemia, phenylketonuria, hemophilia), autoimmune diseases, metabolic disorders (type 2 diabetes), cardiovascular diseases (hypercholesterolemia, myocardial ischemia), fibrosis (hypertrophic scars, liver fibrosis, pulmonary fibrosis, primary sclerosing cholangitis, anemia), etc. [Xu S, Yang K, Li R, Zhang L. mRNA Vaccine Era-Mechanisms, Drug Platform and Clinical Prospection. Int J Mol Sci. 2020 Sep 9;21 (18):6582. doi: 10.3390 / ijms21186582. PMID: 32916818; PMCID: PMC7554980; Qin S, Tang X, Chen Y, Chen K, Fan N, Xiao W, Zheng Q, Li G, Teng Y, Wu M, Song X. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022 May 21;7(1):166. doi: 10.1038 / s41392-022-01007-w. PMID: 35597779; PMCID: РМС9123296].

[0007] In the development of all of the listed drugs, a key task is to create mRNA whose structure will mimic its maturation within the cell. Before being exported from the nucleus to the cytosol for protein translation, eukaryotic RNA undergoes several modifications, the first of which is the addition of a capping structure (cap) to the 5' end of the molecule. This structure ensures efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5'-3' exonucleases, and enables the recognition of foreign RNA. The minimal capping structure is m7GpppNp (7-methylguanosine linked by a 5',5'-triphosphate bridge to the first nucleotide of RNA), also called cap 0. This structure is characteristic of plant and fungal mRNA.For animal mRNA, cap 1 is more typical (it differs from cap 0 by methylation of the first nucleotide of the transcript at the 2'-O-position of ribose) and cap 2 (it differs from cap 0 by methylation of the first and second nucleotides of the transcript at the 2'-O-position of ribose).

[0008] Currently, two methods are used to produce synthetic mRNA: post-transcriptional and cotranscriptional capping. The first method involves the use of vaccinia virus enzymes. This enzymatic complex converts the 5'-triphosphate ends of transcripts in vitro into m7O-cap structures. The vaccinia virus capping system includes three activities (RNA triphosphatase, guanylyl transferase, and guanine X7-methyltransferase), which are necessary for the formation of the complete cap 0 structure (m7Gppp5'N) using GTP and the methyl donor S-adenosylmethionine. Additionally, 2'-O-methyltransferase can be included in the same enzymatic reaction, which leads to the formation of the cap 1 structure by methylating the 2'-O-position of the nucleotide following the cap, which is a natural modification of many eukaryotic mRNAs.

[0009] In cotranscriptional capping, a cap analogue is introduced into the transcription reaction together with nucleoside triphosphates and is incorporated into RNA as the first nucleotide of the synthesized molecule. The most widely used are the standard cap analogue, 7-methylguanosine (m7G), and the symmetrical cap analogue (ARC A), also known as 3'-O-me-7-meGpppG. The standard cap analogue, m7G, can be incorporated into the RNA molecule in both forward and reverse orientations, which significantly reduces the overall translation level of the target protein. ARC A is methylated at the 3' position of m7G, which allows for the insertion of this cap analogue only in the forward orientation and the production of a pool of copied mRNAs with 100% translational activity [A. A. Zagoskin, M. V. Zakharova, M. O. Nagornykh. Structural elements of DNA- and RNA-based vectors for delivering genome editors into higher eukaryotic cells in vitro and in vivo. Molecular Biology, 2022, Vol. 56, No. 6, pp. 1023–1038.

[0010] A number of other synthetic cap analogs with improved properties are also known from the prior art, such as CleanCap, a cap analog from Trilink Biotechnology (USA). The structural formula of this reagent is:

[0011] From patent US10519189B2 a complex is known that includes an initiating copied oligonucleotide primer and a DNA matrix, characterized in that the initiating copied oligonucleotide primer contains the structure:

[0012] wherein B1 and B2 are independently a natural, modified or unnatural nucleoside base; and R1, R2 and R3 are independently OH or O-methyl; characterized in that the DNA template comprises a promoter region comprising a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; and wherein the initiating replicate oligonucleotide primer is hybridized to the DNA template at least at nucleotide positions +1 and +2.

[0013] A trinucleotide analogue of cap is known from patent application US20220002716A, having the formula in which B3 is selected from —OH, halogens, dyes, —ORi, in which Ri is selected from propargyl, tert-butyldimethylsilyl, and a methylene bridge with 4'C; B4 is selected from —OH, dyes, and —OR2, wherein R2 is selected from propargyl and tert-butyldimethylsilyl; or Ri combines with R2 such that B3 and B4 form -2',3'-O-isopropylidine; provided that B3 and B4O6a cannot be —OH. X is selected from —H and —CH3; Bi and B2 are independently selected from adenine, guanine, cytosine, and uracil; R is selected from H linked to a linker of a cell-penetrating peptide, linked to a linker of a cell-penetrating peptide, covalently linked to a dye, and linked to a linker of a dye.

[0014] A trinucleotide analogue of cap is known from patent US11866754B2, having the formula:

[0015] (I) or a stereoisomer, tautomer or salt thereof, in which ring B 1 is in which R1 is C1 alkyl; each of Ra and Rb is H; and Rc is H; ring B2 and ring B3 are selected independently from each other

[0016] where X] is N or N+(R5); R5 is C1-C6 alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, each of which is optionally substituted with one or more substituents selected from the group consisting of C6-C10 aryl, C6-C10 aryloxy, 5- to 10-membered heteroaryl, and 5- to 10-membered heteroaryloxy, each of which is optionally substituted with one or more halo- and cyano-; Rd and Re are each independently H, C1-C6 alkyl, or an amino protecting group, or Rd and R e together with the nitrogen atom to which they are attached, they form a heterocycloalkyl consisting of 4-12 members, -N=CH — R u or — N=N — R u, where RB is phenyl and each of the 4-12 members is heterocycloalkyl, heterocycloalkyl and RB is optionally substituted with one or more substituents selected from OH, halo, oxo, Cl-C6 alkyls, COOH, C(O)O-C1-C6 alkyls, cyano, C1-C6 alkoxides, amino, mono-C1-C6 alkylamino and di-C1-C6 alkylamino; Rf is H, NH2 or C1-C6 alkyl; or Rf and one of Rd and R e together with the two nitrogen atoms to which they are attached and the carbon atom joining the two nitrogen atoms, form a 5- or 6-membered heterocycle which is optionally substituted with one or more of OH, halo, C1-C6 alkyl, C2-C6 alkenyl and C2-C6 alkynyl; R g - is H or methyl; Rh is H or methyl; X2 is O; Y2 is (OR (O) R^m , in which m is 1; R2 is a locked nucleic acid (LNA); R2 is OR3; R3 is H; each of R4 and R4' is OH; each of R 20, R2I, R22, and R23 are —Q3-T3, in which Q3 is a bond and T3 is H; each of R27 and R28 is OR29; and each R29 is H.

[0017] Shanmugasundaram M et al. in their review also describe a number of trinucleotide analogs of cap and the features of their synthesis [Shanmugasundaram M, Senthilvelan A, Kore AR. Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines. Chem Rec. 2022 Aug;22(8):e202200005. doi: 10.1002 / tcr.202200005. Epub 2022 Apr 14. PMID: 35420257; PMCID: PMC9111249].

[0018] Although this field of knowledge is rapidly advancing, all known capped mRNAs have limitations in terms of intracellular stability and in vitro transcription efficiency. Therefore, there remains a need for mRNA cap analogs that can provide high levels of cap efficiency and improved translation efficiency of 5'-capped mRNAs.

[0019] Disclosure of the essence of the invention

[0020] The technical objective of the claimed invention is to expand the arsenal of reagents for capping synthetic mRNA.

[0021] The technical result consists in creating an analogue of the mRNA cap structure, which is included in the mRNA structure cotranscriptionally and ensures improved efficiency of mRNA translation.

[0022] The specified technical result is achieved by creating a trinucleotide capping reagent represented by the formula: where R is H or Me, or its salt acceptable for the transcription reaction.

[0023] In addition, the technical result is achieved by the development of a complex for cotranscriptional capping of mRNA, including a trinucleotide copying reagent according to claim 1 and a DNA matrix, which contains a promoter region containing a transcription start site, having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; wherein the trinucleotide copying reagent according to claim 1 is hybridized with the DNA matrix at least at nucleotide positions +1 and +2.

[0024] A method for producing a trinucleotide copying reagent has also been developed, comprising the following steps:

[0025] (a) obtaining a dinucleotide by liquid-phase oligonucleotide synthesis using the phosphoramidite methodology starting from a 2',3'-protected guanosine derivative and a protected (2',6-dimethyl)adenisylphosphoamidite, followed by 5'-phosphorylation;

[0026] (b) obtaining HA-(3-methoxy)guanosine diphosphate from HA-(3-methoxy)guanosine diol by chemical phosphorylation with phosphorus oxochloride, followed by obtaining the activated imidazolide derivative and condensation with orthophosphate;

[0027] (c) obtaining the P2-imidazolide derivative of KIA-7-methyl-(3-methoxy)guanosine diphosphate by selective methylation with dimethyl sulfate at controlled pH, followed by obtaining the imidazolide derivative with dithiodipyridine;

[0028] (d) obtaining a trinucleotide capping reagent by the reaction of the P2-imidazolide derivative MA-7-methyl-(3-methoxy)guanosine diphosphate with the dinucleotide (2',6-dimethyl)adenosylguanosine-5'-phosphate under the catalysis of divalent metal salts (calcium, magnesium, zinc, cadmium, cobalt).

[0029] The technical result is also achieved by developing the use of a trinucleotide capping reagent for co-transcriptional capping of mRNA.

[0030] Brief description of the drawings

[0031] Fig. 1 shows photographs of mRNA-GFP-transfected cells (A) and control cells (B). Images were taken under a digital inverted microscope in ultraviolet light at 10x magnification.

[0032] Fig. 2 shows the results of luciferase expression assessment in mice 3 hours after intramuscular administration of an mRNA-based vector capped with the developed (mRNA-luc) or known reagent (mRNA-AG-luc) and packaged into lipid particles.

[0033] On the left is a control animal that was injected with a buffer solution.

[0034] In the center is an animal injected with an mRNA-based vector capped with the developed reagent (mRNA-luc), packaged in lipid particles. On the right is an animal injected with an mRNA-based vector replicated with a known reagent (mRNA-AG-luc), packaged in lipid particles.

[0035] A - luminescence intensity ~ from 0.1 * 10 7 relative units up to 0.3* 10 7 rel. units;

[0036] B - luminescence intensity ~ from 0.3* 10 7 relative units up to 0.6* 10 7 rel. units;

[0037] C - luminescence intensity ~ from 0.6* 10 7 relative units up to 0.7* 10 7 rel. units;

[0038] Fig. 3 shows the results of determination of the antibody titer to the S protein of the SARS-CoV-2 virus in animals immunized with a vector based on mRNA capped with the developed (mRNA-luc) or known reagent (mRNA-AG-luc) and packaged in lipid particles.

[0039] The ordinate axis is the titer of antibodies to the S protein of the SARS-CoV-2 virus.

[0040] The abscissa axis is the experimental groups, where

[0041] A - animals immunized with an mRNA-based vector, copied by the developed reagent (mRNA-luc) and packaged in lipid particles.

[0042] B-animals immunized with an mRNA-based vector replicated with a known reagent (mRNA-AG-luc) and packaged into lipid particles.

[0043] * Reliability was calculated using the Mann-Whitney test, p<0.05

[0044] Implementation of the invention

[0045] The synthesis of structurally complex caps, especially on a large scale, is challenging. Multiple functional groups, such as primary and secondary hydroxyl groups in the sugar and the amino group in the nucleoside / nucleotide backbone, make the chemical synthesis of cap analogs extremely challenging. Moreover, these nucleosides / nucleotides are known to degrade under acidic / basic conditions [Kozarski M, Drazkowska K, Bednarczyk M, Warminski M, Jemielity J, Kowalska J. Towards superior mRNA caps accessible by click chemistry: synthesis and translational properties of triazole-bearing oligonucleotide cap analogs. RSC Adv. 2023 Apr 25;13(19):12809-12824. doi: 10.1039 / d3ra00026e. PMID: 37114020; PMCID: PMC 10126820].

[0046] The authors proposed a trinucleotide capping reagent, LNA-m7Gpppm6AmpG, which differs from previously known reagents in that it contains both a closed sugar backbone of a 7-methylguanosine derivative and an adenosine derivative demethylated at positions 6 and 2' at the first nucleotide. The presence of a closed sugar-phosphate backbone increases translation efficiency by enhancing the binding of the transcription factor eIF4E, while methylation at position 6 of the adenosine residue weakens the inhibition of cap-dependent translation under conditions of cellular stress and increases translation efficiency in professional antigen-presenting cells. Modification of the sugar backbone by methylation at the 2' hydroxyl of the 7-methylguanosine residue does not affect translation efficiency but can prevent reagent incorporation in the reverse orientation during in vitro transcription.

[0047] The copying reagent is obtained by the reaction of the β2-imidazolide derivative of LNA-7-methyl-(3-methoxy)guanosine diphosphate with the dinucleotide (2',6-dimethyl)adenisylguanosine-5'-phosphate under the catalysis of divalent metal salts (calcium, magnesium, zinc, cadmium, cobalt). The starting dinucleotide is obtained by liquid-phase oligonucleotide synthesis using the phosphoramidite methodology starting from a 2',3'-protected guanosine derivative and protected (2',6-dimethyl)adenisylphosphoamidite, followed by 5'-phosphorylation. KIA-(3-methoxy)guanosine diphosphate is obtained from LNA-(3-methoxy)guanosine diol by chemical phosphorylation with phosphorus oxochloride, followed by formation of the activated imidazolide derivative and condensation with orthophosphate. The β2-imidazolide derivative LMA-7-methyl-(3-methoxy)guanosine diphosphate is obtained by selective methylation with dimethyl sulfate at controlled pH, followed by formation of the imidazolide derivative with dithiodipyridine.

[0048] The developed copying reagent can be counter-transcriptionally incorporated into the mRNA structure, and it is incorporated only in the direct orientation, which allows obtaining a pool of capped mRNAs with high translational activity.

[0049] The implementation of the invention is disclosed in the following examples.

[0050] Example 1. Preparation of Dmt-2'-Ome-A(6-Me,Bz)-3'-CE phosphoramidite

[0051] Dmt-2'-OMe-A(Bz)-3'-CE phosphoramidite (1 mmol, 888 mg), methyl iodide (4 mmol, 249 μL), and dichloromethane (10 mL) were placed in a single-necked round-bottomed flask equipped with a magnetic stirrer. Simultaneously, a 1 M aqueous sodium hydroxide solution (10 mL) was prepared in a separate beaker. The solution was cooled to room temperature, after which tetrabutylammonium bromide (1 mmol, 322 mg) was added and stirred. The contents of the beaker were transferred to the round-bottomed flask with the reaction mixture. Vigorous stirring of the reaction mixture was continued at room temperature for 1 hour.

[0052] Dichloromethane (100 ml) is then added to the flask, shaken thoroughly, and transferred to a separatory funnel. The organic layer is separated, and the aqueous layer is further extracted with methylene chloride (2 x 50 ml). The organic extracts are combined and dried over anhydrous sodium sulfate for 20-30 minutes. The resulting suspension is filtered and evaporated under reduced pressure at a bath temperature no higher than 35°C.

[0053] The residue after evaporation is purified by flash chromatography on silica gel, eluting with ethyl acetate-toluene-triethylamine (1:5:0.18). Fractions containing the product are collected (TLC control, eluent ethyl acetate-hexane, 2:1, Rf ~ 0.8) and evaporated. The yield of the product after evaporation is 400 mg (44%).

[0054] Example 2. Obtaining a Dmt-protected dinucleotide.

[0055] In a Schlenk flask equipped with a magnetic stirrer, 1 is placed in the form of powders 2-isobutyryl-2',3'-diacetylguanosine (0.75 mmol, 1 equiv) and Dmt-2'-OMe-A(Bz)-3'-CE phosphoramidite (1.13 mmol, 1.5 equiv). The flask with the reagents is evacuated on an oil pump for 40 min. Simultaneously, a solution of tetrazole in acetonitrile (8.5 mL, 4.5 equiv with a concentration of 0.4 M) is prepared in a separate flask under argon atmosphere.

[0056] Fill the Schlenk flask with argon. Next, add the prepared tetrazole solution in acetonitrile under a countercurrent of argon. Close the Schlenk flask, and stir the reaction mixture under an argon atmosphere at room temperature for 2-5 hours until the initial X stain disappears. 2 -isobutyryl-2',3'-diacetylguanosine (TLC control, eluent - dichloromethane-methanol, 100:5, Rf = 0.29).

[0057] After completion of the reaction, a solution of iodine in a pyridine-water mixture (7.5 ml, 4.5 equiv. concentration of 0.45 M, pyridine to water ratio: 98:2) is added and stirring is continued at room temperature for 60-90 minutes (TLC control, eluent - dichloromethane-methanol, 100:5, Rf = 0.37).

[0058] The reaction mixture is diluted with ethyl acetate (120 ml) and transferred to a separatory funnel. The organic layer is washed successively with a saturated solution of Mag3O3 (1 x 50 ml), a saturated solution of NaHCO3 (1 x 50 ml), and a saturated solution of NaCl (2 x 70 ml). The extract is transferred and dried over sodium sulfate for 20 min. The suspension is filtered. The filtrate is collected and evaporated under reduced pressure. The reaction mixture is further evaporated from pyridine using a methanol-toluene mixture (1:1) at a water bath temperature of 42-44°C.

[0059] The residue after evaporation was purified by flash chromatography using a gradient elution system of dichloromethane-methanol (100:1 to 100:7, approx. 1.5 L) on silica gel (50 g). The yield of the final product averaged 85%.

[0060] *H NMR (700 MHz, DMSO-6) 8 12.09 (d, J = 4.8 Hz, 1H); 11.53(s, 1H); 8.56 (d, J = 9.3 Hz, 1H); 8.43 (d, J = 7.2 Hz, 1H); 8.23 (d, J = 4.3 Hz, 1H); 7.35 - 7.26 (m, 5H); 7.24 - 7.13 (m, 9H); 6.80 (ddd, J = 14.7; 8.9; 6.5 Hz; 4H); 6.14 - 6.07 (m, 2H); 5.80 (ddd, J = 24.0; 6.8; 5.8 Hz; 1H); 5.50 (dt, J = 6.0; 3.5 Hz; 1H); 5.20 (q, J = 6.9; 5.3 Hz; 1H); 4.91 (q, J= 6.5 Hz, 1H); 4.49 - 4.33 (m, 4H); 4.26 - 4.14 (m, 2H); 3.71 (d, J = 8.5 Hz, 6H); 3.66 (d, J = 1.0 Hz, 3H); 3.31 - 3.22 (m, 6H); 2.91 - 2.83 (m, 2H); 2.81 - 2.71 (m, 1H); 2.10 (d, J = 11.6 Hz, 3H); 2.01 (d, J= 5.6 Hz, 3H); 1.15 - 1.05 (m, 6H). Example 3. Obtaining a protected dinucleotide.

[0061] The dinucleotide (4.5 mmol) was dissolved in 80% aqueous acetic acid (65 ml, the final dinucleotide concentration was 0.07 M). The reaction mixture was stirred at room temperature for 5 hours (TLC monitoring, eluent - dichloromethane-ethanol, 100:10, R f = 0.32).

[0062] Upon completion, the reaction mixture is evaporated. The reaction mixture is further evaporated from acetic acid and water using a methanol-toluene mixture (1:1) at a water bath temperature of 42-44°C.

[0063] The residue after evaporation was purified by flash chromatography using a gradient elution system of dichloromethane-methanol (100:1 to 100:6, approx. 2 L) on silica gel (80 g). The yield of the final product was 89%.

[0064] *H NMR (700 MHz, DMSO-Дб) 5 12,10 (d, J= 3,6 Hz, 1H), 11,55 (d, J= 2,8 Hz, 1H); 8,71 (d, J= 10,6 Hz, 1H); 8,59 (d, J= 11,2 Hz, 1H); 8,25 (d, J= 4,6 Hz, 1H); 7,37 - 7,31 (m, 3H); 7,26 - 7,20 (m, 2H); 6,11 (ddd, J = 16,8; 6,9; 4,5 Hz, 2H); 5,82 (ddd, J= 11,3; 6,8; 5,9 Hz; 1H); 5,52 (dd, J= 5,9; 3,3 Hz, 1H); 5,31 (t, J= 5,2 Hz, 1H); 5,20 - 5,08 (m, 1H); 4,76 - 4,64 (m, 1H); 4,51 - 4,38 (m, 3H); 4,31 - 4,19 (m, 3H); 3,67 (d, J= 1,6 Hz, 3H); 3,64 - 3,54 (m, 2H); 3,28 (d, J= 35,2 Hz, 3H); 2,97 - 2,90 (m, 2H); 2,76 (hept, J = 6,8 Hz, 1H), 2,12 (d, J = 9,0 Hz, 3H); 2,02 (d, J= 5,8 Hz, 3H), 1,16 - 1,07 (m, 6H).

[0065] Пример 4. Получение защищённого динуклеотида фосфата.

[0066] The dinucleotide (1 equiv, 5.9 mmol) and bis(2-cyanoethyl) diisopropylamidophosphite (2 equiv, 11.8 mmol) are placed in a Schlenk flask equipped with a magnetic stirrer. The flask is evacuated for 15-30 sec and then filled with argon. Simultaneously, a solution of the tetrazole in acetonitrile (60 mL, 4 equiv, with a concentration of 0.4 M) is prepared in a separate flask under argon.

[0067] Next, the prepared tetrazole solution in acetonitrile is added under a countercurrent of argon. The Schlenk flask is sealed, and the reaction mixture is stirred under argon at room temperature for 3-5 hours until the spot of the starting dinucleotide disappears (TLC monitoring, eluent: dichloromethane-methanol, 100:5).

[0068] After the reaction is complete, a solution of iodine in a pyridine-water mixture (60 ml, 4.5 equiv. of 0.45 M concentration, 98:2 ratio) is added and stirring is continued at room temperature for 1 hour (TLC monitoring, eluent - dichloromethane-ethanol, 100:10). Then the reaction mixture is diluted with ethyl acetate (250 ml) and transferred to a separatory funnel. The organic layer is successively washed with a saturated Na2SO solution (1x100 ml), a saturated NaHCCh solution (1x100 ml) and a saturated NaCl solution (2x100 ml). The extract is transferred and dried over sodium sulfate for 20 min. The suspension is filtered. The filtrate is collected and evaporated under reduced pressure. The reaction mixture is evaporated from pyridine using a methanol-toluene mixture (1:1) at a water bath temperature of 42-44°C.

[0069] The residue after evaporation was purified by flash chromatography on a short layer of silica gel (80 g), using a gradient elution system of dichloromethane-methanol (100:2 to 100:10, approx. 2.5 L). The yield of the final product was 70%.

[0070] *H NMR (700 MHz, DMSO-t / b) 5 12.11 (d, J = 3.6 Hz, 1H); 11.56 (d, J = 5.1 Hz, 1H); 8.65 (d, J= 10.9 Hz, 1H); 8.59 (d, J= 10.5 Hz, 1H), 8.25 (d, J= 8.7 Hz, 1H), 7.37 - 7.31 (m, 3H); 7.27 - 7.20 (m, 2H); 6.15 (dd, J = 6.7; 5.4 Hz, 1H); 6.12 (dd, J = 8.0; 6.8 Hz, 1H); 5.84 - 5.77 (m, 1H), 5.54 - 5.49 (m, 1H); 5.33 - 5.23 (m, 1H); 4.84 (dt, = 14.5; 5.8 Hz, 1H); 4.54 - 4.37 (m, 4H); 4.36 - 4.30 (m, 2H); 4.30 - 4.22 (m, 2H); 4.22 - 4.12 (m, 4H); 3.67 (d, J= 2.5 Hz, 3H); 3.30 (d, J= 38.0 Hz, 3H); 2.97 - 2.85 (m, 6H); 2.80 - 2.73 (m, 1H); 2.12 (d, J= 15.5 Hz, 3H); 2.01 (d, J= 5.2 Hz, 3H), 1.12 (dd, J= 8.7; 6.9 Hz, 6H).

[0071] Example 5. Obtaining free dinucleotide phosphate.

[0072] Fully protected dinucleotide phosphate (1 g) is dissolved in 15 ml of a mixture of aqueous ammonia-aqueous methylamine-methanol 3:3:1, placed in an autoclave, heated to 55 °C and maintained at the set temperature for 30 minutes. Then the reaction mixture is evaporated on a rotary evaporator, the residue is coevaporated twice with an equal volume of water, the obtained product is dissolved in a 50 mM solution of triethylammonium bicarbonate to a final concentration of 20 g / l and purified by ion-exchange chromatography on a 50 * 150 HEMA S 1000 DEAE 20i column. Elution is performed in a concentration gradient of triethylammonium bicarbonate 0.1 - 0.7 M in 10 CV with spectrophotometric detection at a wavelength of 254 nm.Fractions containing the target product are evaporated under reduced pressure (5-7 Torr) on a rotary evaporator. The residue is re-evaporated twice with a small volume (approximately 100 ml) of water, then dissolved in a minimal volume of water and a tenfold volume of acetonitrile is added. The resulting mixture is evaporated, the product is suspended in anhydrous acetonitrile, filtered through a 40-porosity Schott filter, and the precipitate is dried under vacuum. The resulting product is further dried over phosphorus pentoxide, yielding the target dinucleotide phosphate as bis triethylammonium salt in 88% yield.

[0073] Example 6. Preparation of LNA-G 5'-monophosphate. To begin the synthesis, a diphosphorylating mixture is prepared. Tributylammonium pyrophosphate (3.29 g, 1 equiv.) is dissolved in acetonitrile (28 mL) in a round-bottomed flask. Tributylamine (8.57 mL, 6 equiv.) is then added. The flask is sealed and shaken thoroughly, observing the formation of a two-phase system. The flask is placed in a freezer (-20°C) and kept for at least 1 hour.

[0074] Then, LNA-guanosine (2 g, 1 equiv), proton sponge (tetramethyl-2,8-diaminonaphthalene, 1.28 g, 1 equiv), and trimethyl phosphate (22 ml) are added to a Schlenk flask equipped with a magnetic stirrer and stirred. The flask is evacuated and then filled with argon. The starting reagents dissolve slowly in trimethyl phosphate at room temperature, so the Schlenk flask is placed in a water bath (50-60°C) and the mixture is allowed to dissolve completely.

[0075] Next, the Schlenk flask is placed in a thermostatically controlled cold water bath (-10°C) and the reaction mixture is stirred for approximately 30 min until cooling. The first portion of phosphorus oxychloride (620 μl, 1.1 equiv) is added under countercurrent argon and stirring is continued for 20 min under the same conditions. Then the second portion of phosphorus oxychloride (390 μl, 0.7 equiv) is added and stirring is continued for 30 min. The diphosphorylating mixture, the preparation of which is described in the first paragraph, is added to the Schlenk flask under countercurrent argon. The remains of the diphosphorylating mixture are washed off the walls of the flask using acetonitrile (10-15 ml). Stirring of the reaction mixture is continued for 30 min under the same conditions.

[0076] Distilled water (170 ml) is added to the reaction mixture and stirred for 40 min at a water bath temperature of -10°C. The reaction mixture is then transferred to a separatory funnel and washed with dichloromethane (4 x 100 ml). The organic (lower) layer is separated and discarded in each batch.

[0077] The resulting aqueous emulsion (upper layer) is transferred to a round-bottomed flask and the residual dichloromethane is removed by evaporation on a rotary evaporator under reduced pressure for 2-3 min until a clear solution is formed at a bath temperature of no more than 40°C. The reaction mixture is transferred to a flat-bottomed flask and neutralized with a saturated aqueous ammonia solution (approximately 800 μl) to pH 6.5. The flask is left stirring at 4°C for 8 hours, then the pH of the reaction mixture is checked; if necessary, an aqueous ammonia solution is added to pH 6.5 (approximately 400 μl). The resulting mixture is chromatographed on DEAE in a water-1 M triethylammonium bicarbonate system with a gradient of 5-50% in 10 column volumes. The target peak at 0.15 M was evaporated and crystallized from acetonitrile to yield 2 g of LNA-guanosine 5'-monophosphate as the triethylammonium salt. Example 7. Preparation of LNA-G 5'-monophosphate imidazolide.

[0078] In a 100 ml Schlonk flask equipped with a stirrer, 1.91 g of LNA-guanosine monophosphate triethylammonium salt, 1 g of imidazole, 1.1 g of dithiodipyridine were placed. The flask was evacuated, then filled with argon, 25 ml of dry DMF were added, then 500 μl of triethylamine, mixed and 1.3 g of dry triphenylphosphine were added. Then the reaction mixture was stirred, complete dissolution of the precipitate was observed within 1 hour. Then the reaction mixture was stirred for 20 hours, then diluted to 200 ml with acetone and centrifuged. The formed precipitate was washed with acetone three times and dried, obtaining 1.7 g (quantitative) of imidazolide as a white powder.

[0079] Example 8. Preparation of LNA-G 5'-diphosphate.

[0080] A 50 ml Schlenk test tube equipped with a stirrer was charged with 1 g of imidazolide LHA-guanosine-5'-monophosphate, evacuated, and filled with argon. Then, 25 ml of dry methylpyrrolidone was added. The mixture was stirred until the precipitate was completely dissolved, after which 700 mg of tributylammonium orthophosphate and 953 mg of anhydrous zinc chloride were added to the reaction mixture. The reaction mixture was stirred for 12 hours. The reaction mixture was diluted with 200 ml of water containing 2.86 g of EDTA and chromatographed on DEAE in a water-1 M triethylammonium bicarbonate system with a gradient of 5-50% in 10 column volumes. The target peak, emerging at 0.4 M, was evaporated and crystallized from acetonitrile to yield 1 g of LNA-guanosine 5'-diphosphate as bis-triethylammonium salt.

[0081] Example 9. Preparation of LNA-m7G 5'-diphosphate.

[0082] A three-necked flask equipped with a stirrer, dropping funnel, and pH electrode was charged with 5.22 g (10 mmol) of LNA-guanosine diphosphate bis-triethylammonium salt, 200 ml of water, and the solution was titrated to pH 4.0 with acetic acid. Next, 15 ml (158 mmol, 15.8 equiv) of dimethyl sulfate were added to the solution, and 150 ml of 1 M sodium hydroxide solution was gradually added with vigorous stirring, maintaining the pH of the solution in the range of 3.8 - 4.2. The addition took approximately 3 hours in total. Then the reaction mass was extracted three times with ethyl acetate (250 ml), the aqueous phase was titrated to pH 6.8-7.0 with a 2M triethylammonium bicarbonate solution, diluted 10 times with water, 1 / 3 of the resulting solution was applied to a Kronlab EcoPlus 50*250 mm column packed with 200 ml of Sobrent HEMA S 1000 DEAE 20um, equilibrated with a 50 tM triethylammonium bicarbonate solution pH 7.6 and chromatographed in a concentration gradient of a 50-600 tM triethylammonium bicarbonate solution in 10 CV.The main peak, appearing at 0.3 M, was collected and pooled. The combined target fraction was evaporated under vacuum at a bath temperature of 38°C, coevaporated twice more with water, then the product was quenched with dry acetonitrile and left to crystallize. The precipitate was filtered on a P4 filter, dried under vacuum, and then under vacuum over phosphorus oxide, yielding 2.04 g (34%) of EHA-7-methylguanosine-5'-diphosphate triethylammonium salt as a white fluffy powder.

[0083] Example 10. Preparation of imidazolide LNA-m7G 5'-diphosphate.

[0084] In a 100 ml Schlonk flask equipped with a stirrer, 2.04 g of LNA-7-methylguanosine-5'-diphosphate triethylammonium salt, 1 g of imidazole, 1.1 g of dithiodipyridine were placed. The flask was evacuated, then filled with argon, 25 ml of dry DMF were added, then 500 μl of triethylamine, mixed and 1.3 g of dry triphenylphosphine were added. Then the reaction mixture was stirred, complete dissolution of the precipitate was observed within 1 hour. Then the reaction mass was stirred for 20 hours, then diluted to 200 ml with acetone and centrifuged. The precipitate was washed with acetone three times and dried, obtaining 1.8 g (quantitative) of imidazolide as a white powder.

[0085] Example 11. Obtaining the copying reagent LNA-m7Gpppm6AmpG.

[0086] A 50 ml Schlenk tube equipped with a stirrer was charged with 1 g of imidazolide E A-7-methylguanosine-5'-diphosphate, evacuated, and filled with argon. Then, 25 ml of dry methylpyrrolidone was added. The mixture was stirred until the precipitate was completely dissolved, after which 645 mg of dinucleotide phosphate and 953 mg of anhydrous zinc chloride were added to the reaction mixture, and the reaction mixture was stirred for 12 hours. The reaction mixture was diluted with 200 ml of water containing 2.86 g of EDTA and chromatographed on DEAE in a water-1 M triethylammonium bicarbonate system with a gradient of 5-60% in 10 column volumes.

[0087] Example 12. Reaction of cotranscriptional capping of mRNA.

[0088] This example demonstrates that the developed trinucleotide copying reagent LNA-m7Gpppm6AmpG can be incorporated into mRNA in vitro. In the first step, several double-stranded DNA templates were designed, each containing genes for a different target protein:

[0089] - the green fluorescent protein (GFP) gene, or

[0090] - luciferase gene (luc), or

[0091] - the SARS-CoV-2 virus S protein gene, as well as all regulatory elements required for target protein gene expression. The DNA was synthesized by Evrogen JSC.

[0092] The DNA molecule was linearized by hydrolysis with the restriction enzyme bspQI (New England Biolabs Inc.). The reaction was carried out according to the manufacturer's recommendations.

[0093] The transcription reaction was performed using 25 μg / ml transcription template, capping buffer, 1000 U / ml mouse RNase inhibitor (New England Biolabs catalog #M0314), 2 U / ml inorganic pyrophosphatase (New England Biolabs catalog #M2403), 4000 units / ml T7 RNA polymerase (New England Biolabs catalog #M0251), 6 mM trinucleotide capping reagent, 1.5 mM GTP, and 7.5 mM each of ATP, CTP, and UTP. One skilled in the art will recognize that other polymerases, such as T7, T3, or SP6 RNA polymerases, can be used instead of T7 RNA polymerase to accomplish the same function using their respective promoters. The reaction mixture was incubated at 37°C for 2 hours. 10 mM Tris-HCl (pH 7.6), 2.5 mM MgCl, 0.5 mM CaCl, and 100 U / ml DNase I (New England Biolabs catalog #M0303) were added to the reaction and incubated for 1 hour at 37°C.The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions. mRNA was eluted in water and dephosphorylated by adjusting the solution to 50 mM bis-Tris-propane HCl (pH 6.0), 1 mM MgCE, 0.1 mM ZnCE and 250 units / mg Antarctic phosphatase (New England Biolabs catalog #M0289). The reaction was incubated at 37°C for 1 hour. The resulting mRNA was purified using the RNeasy Maxi kit (Qiagen catalog #75162) according to the manufacturer's instructions and dissolved in water.

[0094] Thus, as a result of the work carried out, 3 RNA preparations capped with the developed reagent were obtained: mRNA-GFP (the coding sequence of the green fluorescent protein), mRNA-luc (the coding sequence of luciferase), mRNA-S (the coding sequence of the S protein of the SARS-CoV-2 virus).

[0095] At the same time, a series of mRNAs (mRNA-AG-luc, mRNA-AG-S) were obtained, capped with a commercially available reagent - capl analog AG (Biolabmix, according to the manufacturer's instructions) and encoding sequences of luciferase and S protein of the SARS-CoV-2 virus, respectively.

[0096] Example 13. Evaluation of the translational activity of mRNA carrying the green fluorescent protein gene in in vitro experiments.

[0097] The translational activity of mRNA-GFP capped with the developed reagent (see Example 12) was assessed in a human embryonic kidney cell culture (HEK293). HEK293 cells were cultured in DMEM supplemented with 10% FBS, L-glutamine, essential amino acids, and penicillin / streptomycin at 37°C in an atmosphere of 5% CO2. The cells were transfected with 400 ng of mRNA-GFP mixed with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. GFP expression was assessed after 24 h. Figure 1 shows photographs of mRNA-GFP-transfected and control cells. Images were taken under a digital inverted microscope in ultraviolet light at 10x magnification. As can be seen from the presented data, high levels of GFP protein expression are observed in mRNA-GFP-transfected cells.

[0098] Thus, the obtained results indicate that the developed reagent effectively caps mRNA during transcription and ensures its translation.

[0099] Example 14. Packaging of mRNA molecules into lipid particles.

[0100] The mRNA preparations (mRNA-luc, mRNA-S and mRNA-AG-luc, mRNA-AG-S), the preparation of which is described in Example 12, were encapsulated in lipid nanoparticles (LNPs) using a microfluidic mixing process of an mRNA solution (pH 3.0) with a solution of lipids dissolved in alcohol. For this purpose, lipids were dissolved in 96% ethanol at molar ratios of 46.3:9:42.7:1.6 (ionizable lipid (ALC-0315): distearoylphosphatidylcholine: cholesterol: PEGylated lipid (PEG-lipid)). The lipid solution was combined with 10 mM citrate buffer (pH 3.0) containing mRNA (0.2 mg / mL) in a 3:1 volume ratio (aqueous:ethanol) using microfluidic mixing with a Nanoassmblr Benchtop (Precision NanoSystems). The ratio of ionizable nitrogen atoms in the ionizable lipid to the number of phosphate groups in mRNA (N:P ratio) was 6 for each composition.The preparations were dialyzed against PBS (phosphate-buffered saline), pH 7.2, in Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific) for at least 24 hours. The resulting formulations were then sterilized through a 0.22 μm filter and stored at 4°C (PBS) until use. Particle size and zeta potential were measured by dynamic light scattering using a Zetasizer Nano ZS instrument (Malvern Panalytical). mRNA encapsulation was approximately 90% (measured using the RiboGreen assay—Quant-iT™ RiboGreen™ RNA Reagent, Thermo Fisher Scientific).

[0101] As a result of this work, four lipid particle preparations were obtained, containing mRNA copied with the developed reagent (mRNA-luc, mRNA-S) or capped with the known capl analog AG reagent, Biolabmix (mRNA-AG-luc, mRNA-AG-S). The resulting lipid particles were used for in vivo experiments. Example 15. Evaluation of the translational activity of mRNA carrying the luciferin gene in in vivo experiments.

[0102] The aim of this experiment was to evaluate the translational activity of mRNA capped with the developed reagent. mRNA capped with the well-known capl analog AG reagent (Biolabmix) was used as a reference.

[0103] The experiment involved BALB / c mice (18-20g) divided into three groups of five. The animals were administered a single intramuscular injection of the test preparations, consisting of lipid particles containing mRNA (Example 14), at a dose of 10 μg / mouse or phosphate-buffered saline. The following groups of animals were obtained:

[0104] 1) mRNA-luc,

[0105] 2) mRNA-AG-luc,

[0106] 3) phosphate buffered saline.

[0107] Three hours later, mice were injected with 100 μl of a 15 mg / ml solution of the specific luciferase substrate D-luciferin (cat. no. P1041, Promega, USA). Five minutes after substrate administration, bioluminescence was measured using a Lumina II instrument (Perkin Elmer, USA). The data obtained are shown in Fig. 2.

[0108] As can be seen from the experimental results, animals that were injected with mRNA capped with the developed reagent showed higher levels of luminescence compared to mRNA capped with a known reagent.

[0109] Thus, the experimental results show that the developed trinucleotide copying reagent provides improved mRNA translation efficiency.

[0110] Example 16. Immunization of mice with mRNA carrying the SARS-CoV2 Spike protein gene.

[0111] The aim of this experiment was to evaluate the translational activity of mRNA capped with the developed reagent. mRNA capped with the well-known capl analog AG reagent (Biolabmix) was used as a reference.

[0112] The experiment involved BALB / c mice (18-20g) divided into three groups of 10. The animals were injected intramuscularly twice (15 days apart) with the test preparations of lipid particles containing mRNA (Example 14) at a dose of 10 μg / mouse or phosphate-buffered saline. The following groups of animals were obtained:

[0113] 1) mRNA-S,

[0114] 2) mRNA-AG-S, 3) phosphate-buffered saline.

[0115] Blood samples were collected from the animals 14 and 28 days after the first administration of the drug. The serum was then used to determine the titer of binding antibodies.

[0116] The antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) according to the following protocol:

[0117] 1) Protein (S) was adsorbed on the wells of a 96-well ELISA plate for 16 hours at a temperature of +4°C.

[0118] 2) Next, to eliminate non-specific binding, the plate was loaded with 5% milk dissolved in TPBS at a volume of 100 µl per well. The plate was incubated on a shaker at 37°C for 1 hour.

[0119] 3) Using the 2-fold dilution method, serum samples from immunized mice were diluted.

[0120] 4) Add 50 µl of each diluted serum sample to the wells of the plate.

[0121] 5) Next, incubation was carried out for 1 hour at 37°C.

[0122] 6) After incubation, the wells were washed three times with phosphate buffer.

[0123] 7) Then secondary antibodies against mouse immunoglobulins conjugated with horseradish peroxidase were added.

[0124] 8) Next, incubation was carried out for 1 hour at 37°C.

[0125] 9) After incubation, the wells were washed three times with phosphate buffer.

[0126] 10) A solution of tetramethylbenzidine (TMB), which is a substrate for horseradish peroxidase and is converted into a colored compound during the reaction, was then added. The reaction was stopped after 15 minutes by adding sulfuric acid. The optical density (OD) of the solution in each well was then measured using a spectrophotometer at a wavelength of 450 nm.

[0127] The antibody titer was determined as the last dilution in which the optical density of the solution was significantly higher than that of the negative control group. The results (geometric mean) are shown in Fig. 3.

[0128] As can be seen from the presented data, the antibody titer in the group of animals immunized with mRNA capped with the developed trinucleotide copying reagent was significantly higher than in the group of animals immunized with mRNA capped with a known copying reagent. Thus, the results of the experiments demonstrated that mRNA capped with the developed trinucleotide copying reagent exhibits higher translational activity compared to mRNA capped with a known copying reagent.

[0129] Industrial applicability

[0130] All of these examples confirm the ability of the developed trinucleotide copying reagent to be incorporated cotranscriptionally into the mRNA structure and ensure improved mRNA translation efficiency. The developed copying reagent can be used in the production of prophylactic and therapeutic mRNA-based drugs.

Claims

Invention formula 1. A trinucleotide capping reagent represented by the formula: where R is H or Me, or its salt acceptable for the transcription reaction.

2. A complex for cotranscriptional copying of mRNA, comprising a trinucleotide capping reagent according to claim 1 and a DNA template that contains a premotor region containing a transcription start site having a first nucleotide at nucleotide position +1 and a second nucleotide at nucleotide position +2; wherein the trinucleotide capping reagent according to claim 1 is hybridized with the DNA template at least at nucleotide positions +1 and +2.

3. A method for producing a trinucleotide capping reagent according to claim 1, comprising the following steps: (a) obtaining a dinucleotide by liquid-phase oligonucleotide synthesis using the phosphoramidite methodology starting from a 2',3'-protected guanosine derivative and a protected (2',6-dimethyl)adenisylphosphoamidite, followed by 5'-phosphorylation; (b) obtaining 1LCHA-(3-methoxy)guanosine diphosphate from 1LCHA-(3-methoxy)guanosine diol by chemical phosphorylation with phosphorus oxochloride, followed by obtaining the activated imidazolide derivative and condensation with orthophosphate; (c) obtaining the P2-imidazolide derivative 1 of 7-methyl-(3-methoxy)guanosine diphosphate by selective methylation with dimethyl sulfate at controlled pH, followed by obtaining the imidazolide derivative with dithiodipyridine; (d) obtaining a trinucleotide capping reagent by the reaction of the P2-imidazolide derivative L1 A-7-methyl-(3-methoxy)guanosine diphosphate with the dinucleotide (2',6-dimethyl)adenisylguanosine-5'-phosphate under the catalysis of divalent metal salts.

4. The production method according to paragraph 3, wherein the divalent metal salts are selected from calcium, magnesium, zinc, cadmium or cobalt salts.

5. Use of a trinucleotide copying reagent according to paragraph 1 for cotranscriptional copying of mRNA.

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