Capped polynucleotides, capped mRNA, compositions, pharmaceutical proteins, methods of preparation, uses and pharmaceutical formulations

Abstract

The present invention provides capped polynucleotides, capped mRNAs, compositions, pharmaceutical proteins, preparation methods, uses, and pharmaceutical formulations, all of which are in the field of genetic engineering. In the embodiments, capped polynucleotides have at least one pentose linked to the adjacent phosphate via a carbon-carbon double bond. When such capped polynucleotides are used to prepare capped mRNAs, the resulting capped mRNAs are more stable, have better translation activity, and exhibit lower degradation rates, contributing to improved capping rates.

Description

[Technical Field]

[0001] The present disclosure is in the field of genetic engineering and specifically relates to capped polynucleotides, capped mRNA, compositions, pharmaceutical proteins, methods of preparation, uses and pharmaceutical formulations.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This disclosure claims priority to the Chinese application filed on March 13, 2023 with the China Patent Office, bearing application number 202310239920.8 and entitled "Capped Polynucleotides, Capped mRNA, Compositions, Pharmaceutical Proteins, Preparation Methods, Uses and Pharmaceutical Formulations," and the Chinese application filed on March 1, 2024 with application number 202410235630.0 and entitled "Capped Polynucleotides, Capped mRNA, Compositions, Pharmaceutical Proteins, Preparation Methods, Uses and Pharmaceutical Formulations," the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Messenger RNA (mRNA) is a single-stranded polynucleotide chain in the genome that carries protein-coding information and can be used in medicines, vaccines, and other applications. Currently, mRNA can be synthesized outside the body using in vitro transcription (IVT). After mRNA synthesis, the 5' end of the mRNA must be modified; this modification structure is commonly called a cap structure. The cap structure prevents mRNA from being degraded at the 5' end, helps mRNA enter the cytoplasm through a selective channel in the nuclear membrane, enhances translation, and contributes to the entire cleavage process.

[0004] Conventional cap structures generally bind to RNA via a 5',5'-triphosphate bridge (m7GpppN) based on N7-methylguanosine (M7G), and this type of cap structure is called Cap0. However, the capping rate of the Cap0 structure is not high, and the stability of capped mRNA is relatively poor, resulting in a relatively high rate of degradation in vivo. When mRNA with this type of cap structure is used for protein translation, the translation activity is relatively low. Summary of the Invention

[0005] The embodiments of the present disclosure aim to provide capped polynucleotides, capped mRNAs, compositions, pharmaceutical proteins, preparation methods, uses, and pharmaceutical formulations that can improve the stability and capping rate of capped mRNAs, reduce the degradation rate of capped mRNAs in vivo, and improve the translation activity of capped mRNAs.

[0006] In a first aspect, embodiments of the present disclosure provide a capped polynucleotide, wherein the capped polynucleotide is a compound represented by the following structural formula or a salt of a compound represented by the following structural formula: TIFF2026500548000002.tif108114E1 to E5 each represent carbon, Z1 to Z5 each independently represent carbon or oxygen, at least one carbon-carbon double bond exists between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, and between E5 and Z5, B1 to B4 each independently represent a nucleobase, m1 and m2 each represent 0 or 1, n1 is 1, n2 and n3 each independently represent any integer from 0 to 20, G1 and G2 each represent hydrogen or methyl each of R1 to R6 is independently any one of hydrogen, a hydroxyl group, an alkyl group, an alkoxy group, and a halogen; each of X1 to X6 is independently any one of oxygen, sulfur, =NH, and =NCH3; each of Y1 to Y6 is independently any one of oxygen, sulfur, selenium, an aryl group, an alkyl group, an alkoxy group, an aryloxy group, -BH2, -NH, -NCH3, and -N(C(=O)CH3); and each of Q1 and Q2 is independently any one of oxygen, sulfur, and a methylene group.

[0007] As the inventors have discovered, the above technical solution provides a capped polynucleotide of the above structural formula, in which at least one carbon-carbon double bond exists between E1 and Z1, between E2 and Z2, ... and between E5 and Z5. Therefore, by using the above capped polynucleotide to cap the 5' end of an mRNA fragment, the stability and capping rate of the mRNA fragment can be significantly improved, the degradation rate of the capped mRNA fragment in the body can be reduced, and when an mRNA fragment containing the capped polynucleotide is used to translate a protein, the translation activity can be relatively high.

[0008] In one possible embodiment, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, there is a carbon-carbon double bond at least at one position between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, and between E5 and Z5, each of B1 to B4 is independently a nucleic acid base, m1 and m2 are each 0 or 1, n1 is 1, the sum of n1, n2, and n3 is 5 or less, G1 and G2 are each hydrogen or a methyl group, each of R1 to R6 is independently any one of hydrogen, a hydroxyl group, and an alkoxy group, each of X1 to X6 is independently any one of oxygen and sulfur, each of Y1 to Y6 is independently any one of oxygen and sulfur, and Q1 and Q2 are each any one of oxygen and sulfur.

[0009] The above technical proposal can further improve the stability and capping rate of mRNA fragments, further reduce the degradation rate of mRNA fragments in vivo, and further improve the translation activity when translating proteins using mRNA fragments containing the capped polynucleotides.

[0010] In one possible embodiment, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, there is a carbon-carbon double bond at least at one position between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, and between E5 and Z5, each of B1 to B4 is independently a nucleic acid base, n1 is 1, the sum of n1, n2, and n3 is 5 or less, m1 and m2 are each 0 or 1, G1 and G2 are each hydrogen or a methyl group, each of R1 to R6 is independently any one of a hydroxyl group and an alkoxy group, each of X1 to X6 is independently any one of oxygen and sulfur, each of Y1 to Y6 is independently any one of oxygen and sulfur, and Q1 and Q2 are each any one of oxygen and sulfur.

[0011] The above technical proposal can further improve the stability and capping rate of mRNA fragments, further reduce the degradation rate of mRNA fragments in vivo, and further improve the translation activity when translating proteins using mRNA fragments containing the capped polynucleotides.

[0012] In one possible embodiment, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, there is a carbon-carbon double bond at least at one position between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, and between E5 and Z5, each of B1 to B4 is independently a nucleic acid base, m1 and m2 are both 1, n1 is 1, the sum of n1, n2, and n3 is 5 or less, and G1 and G2 are each hydrogen or methyl. each of R1, R4 to R6 is a hydroxyl group, and each of R2 and R3 is an alkoxy group; or each of R2, R4 to R6 is a hydroxyl group, and each of R1 and R3 is an alkoxy group, each of X1 to X6 is independently any one of oxygen and sulfur, each of Y1 to Y6 is independently any one of oxygen and sulfur, and each of Q1 and Q2 is independently any one of oxygen and sulfur.

[0013] As the inventors have found, with the above technical solution, the selection of R1, R2 and R3 can also affect the stability, capping rate and translation activity of the mRNA fragment, which can further improve the stability and capping rate of the mRNA fragment, reduce the degradation rate of the mRNA fragment in the body, and contribute to further improving the translation activity when the mRNA fragment containing the capped polynucleotide is used to translate a protein.

[0014] In one embodiment, the capped polynucleotide is a compound represented by the following structural formula: TIFF2026500548000003.tif5795B1 and B2 are each a nucleic acid base, G1 and G2 are each hydrogen or a methyl group, R1 is a hydroxyl group and R2 is a methoxy group, or R2 is a hydroxyl group and R1 is a methoxy group, X1 to X4 are each independently any one of oxygen and sulfur, Y1 to Y4 are each independently any one of oxygen and sulfur, and Q1 and Q2 are each independently any one of oxygen and sulfur.

[0015] According to the above technical solution, when "R1 is a hydroxyl group and R2 is a methoxy group, or R2 is a hydroxyl group and R1 is a methoxy group," the stability and capping rate of the mRNA fragment are further improved, the degradation rate of the mRNA fragment in the body is further reduced, and the translation activity when the mRNA fragment containing the capped polynucleotide is used to translate a protein is further improved.

[0016] In one embodiment, the capped polynucleotide is a compound represented by the following structural formula: TIFF2026500548000004.tif108112In this structural formula, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, at least one pentose is bonded to the adjacent phosphate group by a carbon-carbon double bond, each of B1 to B4 is independently a nucleobase, n1 is 1, the sum of n1, n2, and n3 is 5 or less, each of R1 to R6 is independently any one of hydrogen, a hydroxyl group, and a methoxy group, G1 and G2 are independently hydrogen or a methyl group, each of R1 to R6 is independently any one of hydrogen, a hydroxyl group, and a methoxy group, and X4 is oxygen or sulfur.

[0017] In one possible embodiment, the carbon-carbon double bond in the capped polynucleotide is at least one of E or Z configuration.

[0018] According to the above technical proposal, due to the presence of carbon-carbon double bonds, capped polynucleotides inevitably exist in cis form (i.e., Z form) and trans form (i.e., E form). As the inventors have found, whether the carbon-carbon double bond is Z form or E form, the stability and capping rate of mRNA fragments can be significantly improved, and the degradation rate of mRNA fragments in the body can be reduced.

[0019] In one possible embodiment, the capped polynucleotide is any one of the following compounds or salts:

[0020] TIFF2026500548000005.tif198169 TIFF2026500548000006.tif208169 TIFF2026500548000007.tif185169 TIFF2026500548000008.tif227169

[0021] Through the above technical solution, the above cap structure can better improve the performance of mRNA fragments.

[0022] In one possible embodiment, the capped polynucleotide is any one of the following compounds or salts:

[0023] TIFF2026500548000009.tif94169

[0024] The above technical proposal can further improve the stability and capping rate of mRNA fragments, further reduce the degradation rate of mRNA fragments in vivo, and further improve the translation activity when translating proteins using mRNA fragments containing the capped polynucleotides.

[0025] In one possible embodiment, the nucleobase includes any one of a natural nucleobase and a modified nucleobase, and optionally the natural nucleobase is any one selected from adenine, uracil, guanine, hypoxanthine, cytosine, thymine, adenine derivatives, uracil derivatives, guanine derivatives, cytosine derivatives, and thymine derivatives, and optionally, in the modified nucleobase, the modifying group is N 6 -methyladenine, N 1 -methyladenine, N 6 -2´-O-dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 1 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, N 1 -methylguanine, and isoguanine.

[0026] In a second aspect, embodiments of the present disclosure provide a composition for preparing capped mRNA, the composition comprising a template DNA and a capped polynucleotide as described above, wherein the template DNA is provided with a transcription position complementary to the capped polynucleotide.

[0027] According to the above technical solution, the above composition can be used to prepare capped mRNA with high stability, low degradation rate, and good translation activity.

[0028] In a third aspect, embodiments of the present disclosure provide a method for preparing capped mRNA, the method comprising performing transcription using a capped mRNA preparation composition according to the second aspect.

[0029] According to the above technical solution and the above preparation method, the capping rate of the capped mRNA can be improved.

[0030] In a fourth aspect, embodiments of the present disclosure provide capped mRNAs that include a primary mRNA fragment and a capped polynucleotide, wherein the primary mRNA fragment is linked to the 5' end of the capped polynucleotide.

[0031] According to the above technical solution, the capped mRNA has better stability, is less likely to be degraded in vivo, and can translate proteins more efficiently.

[0032] In a fifth aspect, embodiments of the present disclosure provide a method for preparing a pharmaceutical protein, the method comprising translating a capped mRNA according to the fourth aspect as a template.

[0033] With the above technical solution, the above pharmaceutical protein preparation method has a relatively high efficiency.

[0034] In a sixth aspect, embodiments of the present disclosure provide a pharmaceutical protein, the pharmaceutical protein being prepared by a method for preparing a pharmaceutical protein according to the fifth aspect.

[0035] In a seventh aspect, embodiments of the present disclosure provide for the use of the above-described capped mRNA in a medicament or vaccine for the treatment of cancer.

[0036] According to the above technical solution, the above mRNA has good stability and is not easily degraded, and when used in the preparation of medicines or vaccines, the medicines or vaccines have good therapeutic and preventive effects.

[0037] In an eighth aspect, embodiments of the present disclosure provide a pharmaceutical formulation comprising the capped mRNA described above and a vector carrying the capped mRNA, wherein the vector comprises any one of a lipid nanoparticle, a liposome, a polymer nanoparticle, a solid lipid nanoparticle, and an emulsion. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 is a schematic diagram showing the effect of differently capped polynucleotides on cell viability. [Figure 2] FIG. 1 is a schematic diagram showing the effect of differently capped polynucleotides on GFP positivity. [Figure 3] FIG. 1 is a schematic diagram showing the effect of different capped polynucleotides on GFP MFI. [Figure 4] FIG. 1 is a schematic diagram showing the effect of differently capped polynucleotides on total GFP expression. [Figure 5] This is an imaging image of a living mouse. [Figure 6] These are images of organs from sacrificed mice. [Figure 7] 1 is a statistical chart of the flux values ​​of organs of mice sacrificed by injecting Fluc LNP (1 μg). [Figure 8] 1 is a statistical chart of the flux values ​​of organs of mice sacrificed by injecting Fluc LNP (10 μg). DETAILED DESCRIPTION OF THE INVENTION

[0039] Hereinafter, the embodiments of the present disclosure will be described in detail using examples. As will be understood by those skilled in the art, the following examples are merely for the purpose of illustrating the present disclosure and are not intended to limit the scope of the present disclosure. In the examples, specific conditions are not specified, but the experiments can be carried out under conventional conditions or under conditions recommended by the manufacturer. For reagents or equipment whose manufacturers are not specified, conventional commercially available products can be used.

[0040] "Alkyl group" refers to an aliphatic hydrocarbon group. The alkyl group portion may be a saturated alkyl group (containing no unsaturated units, such as carbon-carbon double bonds or carbon-carbon triple bonds) or an unsaturated alkyl group (containing at least one unsaturated unit). Whether saturated or unsaturated, the alkyl group portion may be branched or straight-chain. It may contain 1 to 8 carbon atoms (where this is stated, numerical ranges, e.g., "1 to 8," refer to each integer in the specified range; e.g., "1 to 8 carbon atoms" refers to alkyl groups containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms, ... 8 carbon atoms; the present definition includes the appearance of the term "alkyl group" even when no numerical range is specified). Representative alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, etc. "Halogen" refers to fluorine, chlorine, bromine, and iodine.

[0041] In the examples of the present disclosure, the consensus sequence for the most active T7 class III promoters contains a 17-bp sequence upstream and a 6-bp sequence downstream of the transcription start site (Cel1 16:815-25. (1979)). The position of the first transcribed nucleic acid is generally referred to as the +1 transcript nucleotide of the RNA, the second transcribed nucleotide as the +2 transcript nucleotide, and so on.

[0042] The following provides a detailed description of capped polynucleotides, compositions for preparing capped mRNAs, capped mRNAs, pharmaceutical proteins, methods for their preparation, uses, and pharmaceutical formulations according to examples of the present disclosure.

[0043] In an embodiment of the present disclosure, the capped polynucleotide is a compound represented by the following structural formula, or a physiologically acceptable salt of a compound represented by the following structural formula:

[0044] TIFF2026500548000010.tif108125

[0045] In this structural formula, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, and at least one of the bonds between E1 and Z1, E2 and Z2, E3 and Z3, E4 and Z4, and E5 and Z5 is a carbon-carbon double bond. This carbon-carbon double bond structure significantly improves the stability of an mRNA fragment when a capped polynucleotide is used at the 5' end of the mRNA fragment, reducing the degradation rate of the mRNA fragment and improving the translation activity of the mRNA fragment.

[0046] Furthermore, in the above structural formula, each of B1 to B4 is independently a nucleic acid base, m1 and m2 are each 0 or 1, n1 is 1, and each of n2 and n3 is independently an integer between 0 and 20, G1 and G2 are each hydrogen or a methyl group, R1 to R6 are independently any one of hydrogen, a hydroxyl group, an alkyl group (e.g., a long-chain alkyl group), an alkoxy group, and a halogen, or a group that has an electric signal, a magnetic signal, or an optical signal, X1 to X6 are independently any one of oxygen, sulfur, =NH, and =NCH3, Y1 to Y6 are independently any one of oxygen, sulfur, selenium, an aryl group, an alkyl group, an alkoxy group, an aryloxy group, -BH2, -NH, -NCH3, and -N(C(=O)CH3), and Q1 and Q2 are independently any one of oxygen, sulfur, and a methylene group.

[0047] The "nucleobase" in the examples of the present disclosure may be a natural nucleobase or a modified nucleobase. The natural nucleobase is generally any one selected from adenine (A), guanine (G), cytosine (C), uracil (U), thymine (T) and derivatives thereof, and the modified nucleobase is a compound obtained by substituting one or more hydrogen atoms of a natural base with a modifying group, such as N6-methyladenine. In this example, the modifying group is N 6 -methyladenine, N 1 -methyladenine, N 6 -2´-O-dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 1 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, N 1 -methylguanine, and isoguanine.

[0048] In the present disclosure, Y1 to Y6 in the structural formula of a capped polynucleotide of the present disclosure may be negatively charged or uncharged. When Y1 to Y6 in the structural formula of a capped polynucleotide are uncharged, the structural formula of the capped polynucleotide is:

[0049] TIFF2026500548000011.tif111133

[0050] In formula II, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, there is a carbon-carbon double bond at least at one position between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, and between E5 and Z5, each of B1 to B4 is independently a nucleic acid base, m1 and m2 are each 0 or 1, n1 is 1, n2 and n3 are each independently any integer of 0 to 20, G1 and G2 are each hydrogen or a methyl group, and each of R1 to R6 is independently are independently any one of hydrogen, a hydroxyl group, an alkyl group, an alkoxy group, and a halogen; each of X1 to X6 is independently any one of oxygen, sulfur, =NH, and =NCH3; each of Y1 to Y6 is independently any one of a hydroxyl group, a thiol group, selenium, an aryl group, an alkyl group, an alkoxy group, an aryloxy group, -BH3, -NH2, -NHCH3, and -NH(C(=O)CH3); and Q1 and Q2 are independently any one of oxygen, sulfur, and a methylene group.

[0051] When one or more of Y1 to Y6 in the structural formula of the capped polynucleotide of formula II are negatively charged, i.e., when "at least one of Y1 to Y6" in formula II loses a proton H, for example, when "Y1" in formula II becomes "Y1 - " and "Y2" in formula II is "Y2 - " and "Y3" in formula II is "Y3 - " and "Y4" in formula II is "Y4 -" and "Y5" in formula II is "Y5 - " and "Y6" in formula II is "Y6 - ". For example, "Y1" is "Y1 - ", then "Y1 - " is any one of oxygen, sulfur, selenium, an aryl group, an alkyl group, an alkoxy group, an aryloxy group, -BH2, -NH, -NCH3, and -N(C(=O)CH3), and "Y2" is "Y2 - " and "Y3" is "Y3 - " and "Y4" is "Y4 - " and "Y5" is "Y5 - " and / or "Y6" is "Y6 - ", then Y2 - From Y6 - are independently any one of oxygen, sulfur, selenium, an aryl group, an alkyl group, an alkoxy group, an aryloxy group, -BH2, -NH, -NCH3, and -N(C(=O)CH3).

[0052] Formula III in Formula I or Formula II TIFF2026500548000012.tif2128 is a capped polynucleotide TIFF2026500548000013.tif2140, and further comprising a compound of formula IV TIFF2026500548000014.tif2127 It may exist in the form TIFF2026500548000015.tif2241.

[0053] In some alternative embodiments of the present disclosure, in Formula I, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, there is a carbon-carbon double bond at least at one position between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, or between E5 and Z5, each of B1 to B4 is independently a nucleobase, m1 and m2 are each 0 or 1, n1 is 1, the sum of n1, n2, and n3 is 5 or less, G1 and G2 are each hydrogen or a methyl group, R1 to R6 are each independently any one of hydrogen, a hydroxyl group, and an alkoxy group, X1 to X6 are each independently any one of oxygen and sulfur, Y1 to Y6 are each independently any one of oxygen and sulfur, and Q1 and Q2 are each independently any one of oxygen and sulfur.

[0054] According to the above embodiment, the stability and capping rate of the mRNA fragment can be further improved, the degradation rate of the mRNA fragment in the living body can be further reduced, and the translation activity when a protein is translated using the mRNA fragment containing the capped polynucleotide can be further improved.

[0055] Furthermore, in formula I, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, there is a carbon-carbon double bond at least at one position between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, and between E5 and Z5, each of B1 to B4 is independently a nucleobase, n1 is 1, the sum of n1, n2, and n3 is 5 or less, m1 and m2 are each 0 or 1, G1 and G2 are each hydrogen or a methyl group, each of R1 to R6 is independently any one of a hydroxyl group and an alkoxy group, each of X1 to X6 is independently any one of oxygen and sulfur, each of Y1 to Y6 is independently any one of oxygen and sulfur, and Q1 and Q2 are each either one of oxygen and sulfur.

[0056] According to the above embodiment, the stability and capping rate of the mRNA fragment can be further improved, the degradation rate of the mRNA fragment in the living body can be further reduced, and the translation activity when a protein is translated using the mRNA fragment containing the capped polynucleotide can be further improved.

[0057] As the inventors have found, the selection of R1, R2, and R3 can also affect the stability, capping rate, and translation activity of the mRNA fragment. In some alternative embodiments of the present disclosure, in Formula I, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, at least one carbon-carbon double bond exists between E1 and Z1, between E2 and Z2, between E3 and Z3, between E4 and Z4, and between E5 and Z5, each of B1 to B4 is independently a nucleobase, m1 and m2 are both 1, n1 is 1, the sum of n1, n2, and n3 is 5 or less, and G1 and G2 are each each of R1, R4 to R6 is a hydroxyl group, and each of R2 and R3 is an alkoxy group; alternatively, each of R2, R4 to R6 is a hydroxyl group, and each of R1 and R3 is an alkoxy group; each of X1 to X6 is independently any one of oxygen and sulfur; each of Y1 to Y6 is independently any one of oxygen and sulfur; and each of Q1 and Q2 is either any one of oxygen or sulfur.

[0058] The selection of R1, R2, and R3 in the above scheme can further improve the stability and capping rate of the mRNA fragment, reduce the rate of degradation of the mRNA fragment in the body, and contribute to further improving the translation activity when translating a protein using an mRNA fragment containing the capped polynucleotide.

[0059] Furthermore, in some alternative embodiments of the present disclosure, the capped polynucleotide is a compound represented by the following structural formula, or the capped polynucleotide is a salt of a compound represented by the following structural formula:

[0060] TIFF2026500548000016.tif5795

[0061] B1 and B2 are each a nucleic acid base, G1 and G2 are each hydrogen or a methyl group, R1 is a hydroxyl group and R2 is a methoxy group, or R2 is a hydroxyl group and R1 is a methoxy group, X1 to X4 are each independently any one of oxygen and sulfur, Y1 to Y4 are each independently any one of oxygen and sulfur, and Q1 and Q2 are each independently any one of oxygen and sulfur.

[0062] When "R1 is a hydroxyl group and R2 is a methoxy group, or R2 is a hydroxyl group and R1 is a methoxy group," this can further improve the stability and capping rate of the mRNA fragment, further reduce the rate of degradation of the mRNA fragment in the body, and contribute to further improving the translation activity when a protein is translated using an mRNA fragment containing the capped polynucleotide.

[0063] Specifically, in this embodiment, in the capped polynucleotide represented by the structural formula above, G1 and G2 are each hydrogen, m1 and m2 are each 1, X1 to X3 and X5 to X6 are each oxygen, and the entire capped polynucleotide corresponds to the structural formula below or is a salt of a compound represented by the structural formula below.

[0064] TIFF2026500548000017.tif108110

[0065] In this structural formula, X4 is oxygen or sulfur, n1 is 1, the sum of n1, n2, and n3 is 5 or less, each of R1 to R6 is independently hydrogen, a hydroxyl group, or a methoxy group, each of E1 to E5 is carbon, each of Z1 to Z5 is independently carbon or oxygen, and at least one pentose is linked to the adjacent phosphate group by a carbon-carbon double bond. A polynucleotide having a cap corresponding to this structural formula can significantly improve the stability of mRNA fragments, reduce the degradation rate of mRNA fragments, and improve the translation activity of mRNA fragments.

[0066] Since the two structural formulas above contain a carbon-carbon double bond, they inevitably exist in a cis form (i.e., Z form) and a trans form (i.e., E form). As the inventors have discovered, whether the carbon-carbon double bond is Z or E, it can significantly improve the stability and capping rate of mRNA fragments and reduce the degradation rate of mRNA fragments in vivo.

[0067] More specifically, in this example, the capped polynucleotide is any of the structures shown in the table below, or a physiologically acceptable salt of any of the structures shown in the table below.

[0068] TIFF2026500548000018.tif209169 TIFF2026500548000019.tif228169 TIFF2026500548000020.tif218169 TIFF2026500548000021.tif234169 TIFF2026500548000022.tif189169 TIFF2026500548000023.tif178169 TIFF2026500548000024.tif239169 TIFF2026500548000025.tif74169

[0069] Additionally, the capped polynucleotide is any one of the following compounds, or a salt thereof, or a non-salt thereof:

[0070] TIFF2026500548000026.tif85157

[0071] If the capped polynucleotide is selected from compounds having the above structure, it can further improve the stability and capping rate of the mRNA fragment, further reduce the rate of degradation of the mRNA fragment in the body, and further improve the translation activity when translating a protein using an mRNA fragment containing the capped polynucleotide.

[0072] The above capped polynucleotides are prepared by an oligonucleotide solid phase synthesis process, which includes the following steps:

[0073] S100: Pentose containing a vinylphosphonate monomer at the 5' position is used to synthesize N7-methylguanosine 5'-vinyl-5'-diphosphate (m7GDVPIm) intermediate.

[0074] S200: 5'-O-DMT-2'-O-TBDMS phosphoramidite (rA Ac rC Ac , rG dmf , U)), 2'-O-MOE-3'-O-phosphoramidite (A 2’O-TBDMS Ac , C 2’O-MOE Ac , G 2’O-TBDMS dmf , U 2’O-TBDMS)), bis-cyanoethyl-N,N-diisopropyl CED phosphoramidite, etc. are used to synthesize pNpN dinucleotides using a solid-phase synthesizer.

[0075] S300: pNpN dinucleotide is reacted with the phosphate position imidazolium salt N7-methylguanosine 5'-vinyl-5'-diphosphate (m7GVPPIm) to produce the above-mentioned capped polynucleotide.

[0076] In the following examples, the applicant 7 G 2´Ome VpppA 2´Ome pG (abbreviated as 1a) and m 7 G 3´Ome VpppA 2´Ome The synthesis method will be specifically explained using pG (abbreviated as 2a) as an example.

[0077] The capped polynucleotide can be mixed with template DNA to form a composition for preparing capped mRNA. In this case, the template DNA must have a transcription site complementary to the capped polynucleotide, which can then be used in conjunction with the capped polynucleotide to achieve the in vitro transcription process.

[0078] The step of transcribing the capped mRNA comprises the steps of:

[0079] Step 1: Prepare a template DNA so that it contains transcription sites complementary to the corresponding capped polynucleotide.

[0080] The template DNA in this step typically consists of a linearized plasmid, into which the target sequence is inserted, and the target sequence is preceded by a promoter for the corresponding polymerase, such as a T7, T3, or SP6 promoter, followed by bases complementary to the capped polynucleotide as described above, as the transcription site.

[0081] During linearization, the DNA is first linearized by digestion with an appropriate restriction enzyme, followed by purification. Specifically, the restriction enzyme used to linearize the plasmid should be one that can generate blunt ends or 5' overhanging ends (if an enzyme that generates 3' overhanging ends is used to linearize the template, aberrant transcripts will be generated). During purification, the linearized DNA is first treated with phenol-chloroform and then precipitated with ethanol.

[0082] Step 2: Prepare a composition for preparing capped mRNA.

[0083] In this step, the composition typically contains not only the template DNA and the capped polynucleotide, but also an enzyme mixture and a reaction buffer, such as, but not limited to, Tris-HCl (pH 7.9), MgCl, spermidine, DTT, and Triton X-10.

[0084] Step 3: An in vitro transcription reaction is performed to form capped mRNA.

[0085] The capped mRNA thus prepared contains a main mRNA fragment and the above-mentioned capped polynucleotide, with the above-mentioned capped polynucleotide attached to the 5' end of the main mRNA fragment.

[0086] The capped mRNA can be used to prepare medicines for cancer treatment and can be used as a vaccine. For example, the capped mRNA can be carried by a vector, and the vector includes any one of lipid nanoparticles, liposomes, polymer nanoparticles, solid lipid nanoparticles, and emulsions. The capped mRNA can also be used to prepare pharmaceutical proteins, for example, by translating the capped mRNA as a template to obtain pharmaceutical proteins.

[0087] The features and performance of the present disclosure will be described in more detail below with reference to examples.

[0088] Example 1 This example provides a capped polynucleotide, designated 1a. 1a was prepared by reacting intermediate 1a-6 with intermediate 1a-8. During the preparation process, several intermediates were characterized using nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry, respectively. The specific pathways and steps are as follows:

[0089] (1) The preparation route of intermediate 1a-6 was as follows.

[0090] TIFF2026500548000027.tif73156

[0091] The specific reaction process was as follows:

[0092] (1.1) Under nitrogen gas protection, 7.50 g of compound 1a-1 (1 equivalent) was dissolved in 120 mL of DMF (N,N-dimethylformamide). The reaction mixture was cooled to 0°C, and 7.2 mL of 2,6-dimethylpyridine and 24.5 mL of TMSBr (bromotrimethylsilane) were added. The reaction mixture was then heated to 50°C. After stirring for 12 hours, the mixture was cooled to room temperature and quenched by adding 120 mL of methanol. Then, 120 mL of methanol / aqueous ammonia (v:v=1 / 1) was added to the reaction mixture, and the mixture was heated to 45°C and stirred for 3 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure and dried. The crude product was purified by C18 preparative chromatography to obtain compound 1a-3 (5.00 g, 89% yield). Nuclear magnetic resonance spectroscopy data for 1a-1: 31 PNMR (243 MHz, CDCl3) δ (ppm): 7.98 (s), liquid chromatograph mass spectrometer data: ESI-MS: m / z 373.1, [M+H] + =374.2.

[0093] (1.2) Under nitrogen gas protection, 4.8 g of compound 1a-3 (1 equivalent) was dissolved in 480 mL of tert-butyl alcohol / water (v:v = 1 / 1), and 0.1 mol / L hydrochloric acid was added to adjust the pH to 2.0. 3.68 g of morpholine (3 equivalents) was added, and the mixture was stirred at 25 °C for 15 minutes, then heated to 80 °C and refluxed. 8.60 g of DCC (Dicyclohexylcarbodiimide, N,N'-dicyclohexylcarbodiimide, 3 equivalents) was dissolved in 24 mL of tert-butyl alcohol and added to the reaction solution in three portions. The mixture was refluxed at 80 °C for 5 hours. The mixture was cooled to room temperature, filtered, and the organic phase was concentrated under reduced pressure to dryness. The crude product was redissolved in 100 mL of water and extracted with 100 mL of DCM (dichloromethane). The aqueous phase was concentrated to dryness to obtain the crude product of compound 1a-4, which did not require purification and was used directly in the next step.

[0094] (1.3) Compound 1a-4 was dissolved in 100 mL of ethanol / acetonitrile (v:v=1 / 1), concentrated under reduced pressure to remove water, and redissolved in 50 mL of DMSO (dimethylsulfoxide). 17.6 g of triethylamine phosphate (4 equivalents) was added, and the mixture was stirred at 30°C for 12 hours. The temperature was then lowered to 0°C, and 200 mL of water was added to quench the reaction. The reaction solution was purified with a chlorine-based resin to obtain compound 1a-5 (2.91 g, two-step yield, 50%). Liquid chromatograph mass spectrometry data for 1a-5: ESI-MS: m / z 453.0, [M+H] + =453.1.

[0095] (1.4) Under nitrogen gas protection, 2.91 g of compound 1a-5 (1 equivalent) was dissolved in 30 mL of water, the pH was adjusted to 4.3 with acetic acid, 0.81 g of dimethyl sulfate (6 equivalents) was added, and the mixture was stirred at 25 °C for 20 hours. The reaction mixture was extracted once with 50 mL of DCM, and the aqueous phase was purified with a chlorine-based resin to give compound 1a-6 (2.16 g, yield: 72%).

[0096] Hydrogen nuclear magnetic resonance spectrum parameters of 1a-6: 1 H NMR (500 MHz, DO) δ (ppm): 9.01 (s, 1H), 6.66-6.57 (m, 1H), 6.19 (t, 1H, J = 20.0 Hz), 6.07 (d, 1H, J = 3.50 Hz), 4.63 (m, 1H), 4.41-4.37 (m, 2H), 4.04 (s, 3H), 3.64 (s, 3H). Phosphorus nuclear magnetic resonance spectral parameters: 31 P NMR(243MHz,D2O)δ(ppm):2.67(d,1P,P α ),-11.17(d,1P,P β Liquid chromatograph mass spectrometer data: ESI-MS: m / z 467.1, [M+H] + =468.3.

[0097] (2) The preparation route of intermediate 1a-8 was as follows.

[0098] TIFF2026500548000028.tif49135

[0099] The specific reaction process was as follows:

[0100] Under nitrogen gas protection, 20.0 g of compound 1a-7 (1 equivalent) was dissolved in 300 mL of DMSO, and the reaction mixture was heated to 38 ° C. 3.0 g of imidazole (10 equivalents), 4.0 g of 2,2-dithiodipyridine (3 equivalents), 4.8 g of triphenylphosphine (3 equivalents), and 1.9 mL of triethylamine (3 equivalents) were added. The reaction mixture was stirred at 38 ° C. for 2 hours, and then added to an acetone solution (3000 mL, containing 2% sodium perchlorate, w / v), stirred for 1 hour, filtered, and the solid was concentrated under reduced pressure to obtain compound 1a-8 (18 g, yield: 90%). Liquid chromatographic mass spectrometry data for 1a-8: ESI-MS: m / z 756.2, [M+H] + =757.3.

[0101] (3) 1a was prepared using 1a-6 and 1a-8, and the reaction pathway was as follows:

[0102] TIFF2026500548000029.tif62150

[0103] The specific reaction process was as follows: Under nitrogen gas protection, 1g of compound 1a-8 (1 equivalent) and 2g of compound 1a-6 (2.3 equivalents) were dissolved in 40mL of DMSO, 1g of anhydrous magnesium chloride (8 equivalents) was added, and the mixture was stirred at 25°C for 2 hours. The reaction solution was added to 200mL of H2O, and the reaction solution was filtered and purified with chlorine-based resin to obtain crude compound 1a, which was further purified by C18 preparative chromatography to obtain compound 1a (0.5g, yield: 16%). Hydrogen nuclear magnetic resonance spectrum data of 1a: 1H NMR(500MHz,D2O)δ(ppm):8.77(s,1H),8.25(s,1H),7.99(s,1H),7.84(s,1H),6.72-6.68(m,1H),6.64-6.55(m,1H),6.29-6.19(m,1H),5. 87(d,1H,J=4.0Hz),5.73(t,2H,J=4.0Hz),4.40-4.38(m,3H),4.30-4.28(m,3H),4.12-4.07(m,6H),3.92(s,3H),3.42(s,3H),3.32(s,3H). Phosphorus nuclear magnetic resonance spectrum data: 31 P NMR(243MHz,D2O)δ(ppm):3.92(d,1P,P α ),-11.73(m,1P,P β ),-23.14(q,1P,P γ ), -0.92(s,1P). Liquid chromatograph mass spectrometer data: ESI-MS: m / z 1155.2, [M+H] + =1155.4.

[0104] Example 2 This example provides a capped polynucleotide, designated 2a. 2a was prepared by reacting intermediate 2a-11 with intermediate 2a-13. During the preparation process, several intermediates were characterized using a nuclear magnetic resonance phosphorus spectrometer and a liquid chromatography mass spectrometer, respectively. The specific pathways and steps are as follows:

[0105] 1. The preparation route of intermediate 2a-11 was as follows:

[0106] TIFF2026500548000030.tif151157

[0107] The specific reaction process was as follows:

[0108] (1) Under nitrogen gas protection, 5 g of compound 2a-1 (1.0 equivalent) was dissolved in 100 mL of DMF, and 10 mL of pyridine was added. The temperature of the reaction solution was lowered to 0°C, and 3.8 g of TBDMS-Cl (tert-butyldimethylsilyl chloride, tert-butyldimethylchlorosilane) was added. The temperature was raised to 15°C and the reaction was carried out for 20 hours. After the reaction was completed, 2.1 g of sodium bicarbonate and 10 mL of methanol were added to quench the reaction. The reaction was then extracted once with a 5% aqueous sodium bicarbonate solution and ethyl acetate. The aqueous phase was extracted three times with ethyl acetate. The organic phases were combined and extracted three times with a 10% aqueous sodium chloride solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 7.4 g of crude compound 2a-2.

[0109] (2) The crude product of compound 2a-2 was dissolved in 74 mL of pyridine, and 1.1 g of DMAP (4-dimethylaminopyridine, 0.5 equivalents) and 7.1 g of acetic anhydride (4 equivalents) were added. The mixture was heated to 40 °C and reacted for 10 hours. The reaction was then stopped. After the temperature was lowered to room temperature, 5% aqueous sodium bicarbonate was added to the mixture until no more bubbles were generated. The mixture was extracted once with dichloromethane. The organic phase was separated, extracted once with 5% aqueous sodium bicarbonate, and then extracted once with 10% aqueous sodium chloride. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 7.1 g of crude compound 2a-3.

[0110] (3) The crude product of compound 2a-3 was dissolved in 71 ml of pyridine, and 2.1 g of HF·Py (pyridine hydrofluoride) was added at 0°C. The mixture was heated to 35°C and reacted for 24 hours. The reaction was stopped, and the pyridine was removed by concentration under high vacuum. 5 g of crude product of compound 2a-4 was obtained by concentration.

[0111] (4) Under nitrogen gas protection, the crude product of compound 2a-4 was dissolved in 50 ml of DMSO, and 8.1 g of DCC (3 equivalents) was added. Then, 1.03 g of pyridine (1 equivalent) and 0.75 g of trifluoroacetic acid (0.5 equivalents) were added and dissolved. The reaction mixture was reacted at 25 ± 5 ° C for 2.5 hours, filtered, and the reaction mixture was extracted and separated with ethyl acetate and water. The lower aqueous phase was extracted once with ethyl acetate. The organic phases were combined, washed once with saturated aqueous sodium chloride, and then separated. The upper organic phase was dried over anhydrous sodium sulfate. The mixture was then filtered and concentrated to obtain 5 g of crude compound 2a-5.

[0112] (5) Under nitrogen gas protection, 5.67 g of tetraethyl methylenediphosphonate (1.5 equivalents) was dissolved in 50 ml of tetrahydrofuran, cooled to -60±5°C, and 5.67 g of sodium tert-pentoxide (1.6 equivalents) was added and stirred at -60±5°C for 1 hour. 5 g of the crude product of compound 2a-5 was dissolved in 50 ml of tetrahydrofuran and added dropwise to the above reaction system, controlling the temperature at -60±5°C during the dropping process. After completion of the reaction, the mixture was stirred at -60±5°C for an additional hour, then slowly warmed to room temperature and stirred for an additional 8 hours. After the reaction was complete, saturated NH4Cl solution was added and stirred for 10-15 minutes. Ethyl acetate was added and the mixture was extracted and separated. The lower aqueous phase was extracted once more with ethyl acetate. The upper organic phases were combined and washed once with saturated NH4Cl and saturated NaCl solutions, respectively. The upper organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated to give 4.3 g of crude product. The crude product was purified by C18 preparative chromatography to give compound 2a-6 (2.00 g, overall yield: 23%). ESI-MS: m / z 513.2, [M+H] + =514.3.

[0113] (6) Under nitrogen gas protection, 10 g of compound 2a-6 (1 equivalent) was dissolved in 150 mL of DMF. The reaction mixture was cooled to 0 °C, and 9.36 mL of 2,6-dimethylpyridine and 31.85 mL of TMSBr were added. The reaction mixture was then heated to 50 °C. After stirring for 12 hours, the mixture was cooled to room temperature and quenched by adding 150 mL of methanol. Then, 150 mL of methanol / aqueous ammonia (v / v = 1 / 1) was added to the reaction mixture, and the mixture was heated to 45 °C and stirred for 3 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure and dried. The crude product was purified by C18 preparative chromatography to obtain compound 2a-8 (5.81 g, yield: 80%). Phosphorus nuclear magnetic resonance spectral data for 2a-8: 31 P NMR (243 MHz, CDCl3) δ (ppm): 7.91 (s). Liquid chromatograph mass spectrometer data: ESI-MS: m / z 373.1, [M+H] + =374.2.

[0114] (7) Under nitrogen gas protection, 5 g of compound 2a-8 (1 equivalent) was dissolved in 500 mL of tert-butyl alcohol / water (v / v = 1 / 1), and 0.1 mol / L hydrochloric acid was added to adjust the pH to 2.0. 3.83 g of morpholine (3 equivalents) was added, and the mixture was stirred at 25 °C for 15 minutes. The mixture was then heated to 80 °C and refluxed. 8.96 g of DCC (3.0 equivalents) was dissolved in 25 mL of tert-butyl alcohol and added in three portions to the reaction mixture. The mixture was refluxed at 80 °C for 5 hours. The mixture was cooled to room temperature, filtered, and the organic phase was concentrated to dryness under reduced pressure. The crude product was redissolved in 100 mL of water, extracted with 100 mL of DCM, and the aqueous phase was concentrated to dryness to obtain crude compound 2a-9, which was used directly in the next step without further purification.

[0115] (8) Compound 2a-9 was dissolved in 100 mL of ethanol / acetonitrile (v / v = 1:1), concentrated under reduced pressure to remove water, and redissolved in 50 mL of DMSO. 18.3 g of triethylamine phosphate (4 equivalents) was added, and the mixture was stirred at 30 °C for 12 hours. The temperature was then lowered to 0 °C, and 200 mL of water was added to quench the reaction. The reaction solution was purified with a chlorine-based resin to obtain compound 2a-10 (3.0 g, two-step yield, 51%). Liquid chromatograph mass spectrometer data for 2a-10: ESI-MS: m / z 453.0, [M+H] + =453.1.

[0116] (9) Under nitrogen gas protection, 3.0 g of compound 2a-10 (1 equivalent) was dissolved in 30 mL of water, the pH was adjusted to 4-4.5 with acetic acid, 0.84 g of dimethyl sulfate (6 equivalents) was added, and the mixture was stirred at 25°C for 20 hours. The reaction mixture was extracted once with 50 mL of DCM, and the aqueous phase was purified with a chlorine-based resin to obtain compound 2a-11 (2.5 g, yield: 80%). Proton nuclear magnetic resonance spectral data for 2a-11: 1 H NMR(500MHz,D2O)δ(ppm):9.12(s,1H),6.59-6.42(m,1H),6.05(t,1H,J=20.0Hz), 5.91(d,1H,J=3.50Hz),4.50(m,1H),4.35-4.28(m,2H),3.95(s,3H),3.59(s,3H). Phosphorus nuclear magnetic resonance spectrum data: 31 P NMR(243MHz,D2O)δ(ppm):2.61(d,1P,P α ),-11.10(d,1P,P β Liquid chromatograph mass spectrometer data: ESI-MS: m / z 467.1, [M+H]+ = 468.3.

[0117] 2. The preparation route of intermediate 2a-13 was as follows:

[0118] TIFF2026500548000031.tif58157

[0119] The specific reaction process was as follows. Under nitrogen gas protection, 20.0 g of compound 2a-12 (1 equivalent) was dissolved in 300 mL of DMSO. The reaction mixture was heated to 38 °C, and 3.0 g of imidazole (10 equivalents), 4.0 g of 2,2-dithiodipyridine (3 equivalents), 4.8 g of triphenylphosphine (3 equivalents), and 1.9 mL of triethylamine (3 equivalents) were added. The reaction mixture was stirred at 38 °C for 2 hours, and then added to an acetone solution (3000 mL, containing 2% sodium perchlorate, w / v), stirred for 1 hour, filtered, and the solid was concentrated under reduced pressure to obtain compound 2a-13 (18 g, yield: 90%). Liquid chromatograph mass spectrometry data for 2a-13: ESI-MS: m / z 756.2, [M+H] + =757.3.

[0120] 3. 2a was prepared using 2a-9 and 2a-13, and the reaction pathway was as follows:

[0121] TIFF2026500548000032.tif61148

[0122] The specific reaction process was as follows: Under nitrogen gas protection, 1g of compound 2a-13 (1 equivalent) and 2g of compound 2a-11 (2.3 equivalents) were dissolved in 40mL of DMSO, 1g of anhydrous magnesium chloride (8 equivalents) was added, and the mixture was stirred at 25°C for 2 hours. The reaction solution was added to 200mL of HO, and the reaction solution was filtered and purified with chlorine-based resin to obtain crude compound 2a, which was further purified by C18 preparative chromatography to obtain compound 2a (0.5g, yield: 16%). Hydrogen nuclear magnetic resonance spectrum data of 2a: 1H NMR(500MHz,D2O)δ(ppm):8.9(s,1H),8.45(s,1H),7.79(s,1H),7.70(s,1H),6.75-6.61(m,1H),6.61-6.50(m,1H),6.19-6.08(m,1H),5.8 9(d,1H,J=4.0Hz),5.69(t,2H,J=4.0Hz),4.43-4.40(m,3H),4.33-4.30(m,3H),4.10-4.08(m,6H),3.95(s,3H),3.38(s,3H),3.30(s,3H). Phosphorus nuclear magnetic resonance spectrum data: 31 P NMR(243MHz,D2O)δ(ppm):3.90(d,1P,P α ),-11.73(m,1P,P β ),-23.14(q,1P,P γ ), -0.92(s,1P). Liquid chromatograph mass spectrometer data: ESI-MS: m / z 1155.2, [M+H] + =1155.4.

[0123] Experimental example 1: Detecting the cap rate First, a composition for preparing capped Fluc mRNA (i.e., an IVT reaction system) was prepared: 5 mM NTP (nucleoside triphosphate), 5 mM of the target capped polynucleotide (the capped polynucleotides of Examples 1 and 2 and the capped polynucleotides of Comparative Example), 1X buffer, 1 μg / μL of EGFP (enhanced green fluorescent protein) linearized template, and 20 KU / mL of the enzyme mixture.

[0124] The steps for preparing a 10X buffer solution were as follows: Tris-HCl (pH 7.9), MgCl, spermidine, and DTT were mixed to prepare a 10X buffer solution containing magnesium ions. In the buffer solution, the Tris-HCl concentration was 400 mM, the MgCl concentration was 200 mM, the spermidine concentration was 20 mM, and the DTT concentration was 100 mM.

[0125] The mixed enzyme system was prepared as follows: T7 RNA polymerase, inorganic pyrophosphatase, and ribonuclease inhibitor were mixed to prepare the mixed enzyme system. In the mixed enzyme system, the T7 RNA polymerase concentration was 400 U / μL, the inorganic pyrophosphatase concentration was 0.1 U / μL, and the nuclease inhibitor concentration was 20 U / μL.

[0126] The IVT reaction system was incubated at 37°C for 3 hours. After Dnase I digested the template, the Fluc mRNA was precipitated with LiCl and purified to obtain the Fluc mRNA product. NTPs were ATP, GTP, CTP, or uridine (uridine triphosphate (i.e., UTP) or N1-methyl-pseudouridine sodium solution (i.e., N1-Me-pUTP).

[0127] The reaction system was divided into four groups based on the capped polynucleotide and NTP, and the capping rate was measured. The measurement method was as follows: (1) Annealing of probe and mRNA: 500 pmol of probe and 100 pmol of the above-mentioned Fluc mRNA product were added to a 1x RNase H reaction buffer, mixed uniformly, and then annealed (maintained at 95°C for 5 minutes, then cooled to 40°C at 2°C / min) to obtain a reaction solution. (2) RNase H enzyme digestion and elution: 100 μL of magnetic beads (Dynabeads MyOne Streptavidin C1, Invitrogen #65002) were taken and washed twice with wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl), then the above reaction mixture was added and mixed uniformly. 10 μL of RNase H enzyme (M0297L) was added and reacted at 37°C for 4 hours. After the reaction was complete, the beads were washed twice with wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl), 50 μL of enzyme-free water was added, and the mixture was heated to 80°C and incubated for 3 minutes. The centrifuge tube was then placed on a magnetic stand to elute the supernatant. The eluted mixture was analyzed by liquid chromatography-mass spectrometry using a Waters reverse-phase C18 column (2.1 × 50 mm). Fluid phase A: 200 mM hexafluoroisopropanol and 8.15 mM triethylamine, pH 7.9; Fluid phase B: methanol. Integration yields yielded two distinct sequences based on molecular weight. One sequence contained a 5'-capped structure, while the other contained no 5'-capped structure. The capping rate (%) was calculated as (M1 / M2) × 100%, where M1 is the sum of the mass spectrometry response intensities of all sequences containing a 5'-capped structure, and M2 is the sum of the mass spectrometry response intensities of all sequences containing a 5'-capped structure and all sequences without a 5'-capped structure. The yield and capping rate of Fluc mRNA are shown in Table 1.In the above system, "mM" stands for "mmol / L."

[0128] TIFF2026500548000033.tif107163

[0129] The structural formula of the comparative example was as follows:

[0130] TIFF2026500548000034.tif5288

[0131] As can be seen from Table 1, compared to the polynucleotides capped according to the comparative example, the polynucleotides capped according to Examples 1 and 2 of the present disclosure can improve the capping rate of mRNA.

[0132] Experimental example 2: Translation performance test The translation performance of each of the Fluc mRNA products of Groups 1 to 6 in Experimental Example 1 was measured.

[0133] Test method for translation performance: The Fluc mRNA synthesized in Groups 1 to 6 in Experimental Example 1 was transfected into JAWS II cells, a mouse DC cell line. 5Cells were seeded into 24-well plates at a density of 1 μg / well and transfection was performed when the cell density was approximately 80%. Each well was transfected with 1 μg of Fluc mRNA. The transfection reagent was Lipofectamine MessengerMAX Transfection Reagent (Invitrogen), and the transfection steps were performed according to the manufacturer's instructions. To investigate the stability of different cap structures under pressure, this example further treated the cells with 5000 U / mL of IFNα (i.e., interferon α) for 6 hours before performing the transfection experiment. After 24 and 72 hours of transfection, cells were sampled and subjected to flow cytometry to detect cell activity and GFP expression. The results are shown in Tables 2 to 5 and Figures 1 to 4.

[0134] TIFF2026500548000035.tif113169

[0135] TIFF2026500548000036.tif108169

[0136] TIFF2026500548000037.tif108169

[0137] TIFF2026500548000038.tif107169

[0138] In Tables 2 to 5, "MFI" represents the mean fluorescence intensity, and "Total GFP" represents the total fluorescence signal.

[0139] The abscissas 1 to 6 in Figures 1 to 4 represent Group 1 to Group 6, respectively. In Figures 1 to 4, "Ctrl-24h" represents the experimental group without IFNα treatment and transfection time of 24 hours, "Ctrl-72h" represents the experimental group without IFNα treatment and transfection time of 72 hours, "IFNα-24h" represents the experimental group with IFNα treatment for 6 hours followed by transfection for 24 hours, and "IFNα-72h" represents the experimental group with IFNα treatment for 6 hours followed by transfection for 72 hours.

[0140] As can be seen from Tables 2 to 5 and Figures 1 to 4, neither mRNA prepared with the capped polynucleotides of the Comparative Example nor those of Examples 1 and 2 impaired cell viability. Compared to mRNA prepared with the capped polynucleotides of the Comparative Example, mRNA prepared with the capped polynucleotides of Examples 1 and 2 enhanced GFP expression. Furthermore, after treatment with IFNα, GFP expression in mRNA prepared with the capped polynucleotides of the Comparative Example was significantly impaired, whereas GFP expression in mRNA prepared with the capped polynucleotides of Examples 1 and 2 showed relatively little change. Therefore, compared to mRNA prepared with the capped polynucleotides of the Comparative Example, mRNA prepared with the capped polynucleotides of Examples 1 and 2 exhibited higher stability and better translation efficiency.

[0141] Experimental example 3: Translation performance test Using a PNI ignite instrument, each of the Fluc mRNAs prepared in Groups 1 to 6 in Experimental Example 1 above was encapsulated at a flow rate of 3:1 between aqueous and ethanol phases. The molar ratio of each liposome component in the ethanol phase was Dlin-MC3:PEG2000-DMG:DSPC:cholesterol = 50:1.5:38.5:10. After encapsulation, the corresponding PBS solution was substituted and concentrated using a 100K molecular weight ultrafiltration centrifuge tube to obtain 0.2 mg / mL Fluc LNP.

[0142] Using a PNI ignite instrument, encapsulation was performed at a flow rate of 3:1 between the aqueous and ethanol phases, with the molar ratio of each liposome component in the ethanol phase being Dlin-MC3:PEG2000-DMG:DSPC:Cholesterol = 50:1.5:38.5:10. After encapsulation, the solution was substituted with PBS solution using a 100K molecular weight ultrafiltration centrifuge tube and concentrated to obtain a 0.2 mg / mL blank LNP.

[0143] The prepared Fluc LNPs (1 μg and 10 μg doses) and blank LNPs (50 μL dose) were intravenously injected into C57 mice. Six hours after injection, live imaging was performed. The mice were then sacrificed, and their organs (lungs, liver, spleen, and lymph nodes) were imaged using an imaging device, and the flux values ​​were calculated. The live mouse imaging results are shown in Figure 5, and images of the organs of sacrificed mice are shown in Figure 6. The flux values ​​of the organs of mice injected with Fluc NPs (1 μg dose) and sacrificed are shown in Figure 7, and the flux values ​​of the organs of mice injected with Fluc NPs (10 μg dose) and sacrificed are shown in Figure 8.

[0144] In Figures 5 and 6, numbers 1 to 6 represent Fluc NPs (injection dose 1 μg) corresponding to Groups 1 to 6 in Experimental Example 1, respectively; number 7 represents blank LNPs (injection volume 50 μL); and numbers 8 to 13 represent Fluc NPs (injection dose 10 μg) corresponding to Groups 1 to 6 in Experimental Example 1, respectively. In Figure 6, the organs in each image are, from top to bottom, the lung, liver, spleen, and lymph nodes. In Figures 7 and 8, the ordinate "Total Flux" represents the fluorescence value, Whole represents the entire organ, Lung represents the lung, Liver represents the liver, Spleen represents the spleen, and LN represents the lymph nodes.

[0145] As can be seen from Figures 5 to 8, the translation efficiency of the mRNA prepared with the capped polynucleotides of Examples 1 and 2 is better than that of the mRNA prepared with the capped polynucleotides of the comparative example. [Industrial Applicability]

[0146] The capped polynucleotides, capped mRNAs, compositions, pharmaceutical proteins, preparation methods, uses and pharmaceutical formulations of the present disclosure can improve the stability and capping rate of capped mRNAs, reduce the degradation rate of capped mRNAs in vivo, and improve the translation activity of capped mRNAs.

Claims

1. 1. A capped polynucleotide comprising: The capped polynucleotide is a compound represented by the following structural formula: E 1 From E 5 Each of Z is a carbon; 1 From Z 5 each independently is carbon or oxygen; 1 and Z 1 Between E 2 and Z 2 Between E 3 and Z 3 Between E 4 and Z 4 Between E 5 and Z 5 and at least one carbon-carbon double bond is present between B 1 From B 4 each of which is independently a nucleobase, m 1 , m 2 are each 0 or 1, n 1 is 1, and n 2 , n 3 are each independently any integer from 0 to 20; G 1 , G 2 are each hydrogen or a methyl group, R 1 From R 6 each independently represents one of hydrogen, a hydroxyl group, an alkyl group, an alkoxy group, and a halogen; X 1 From X 6 each independently represents oxygen, sulfur, ═NH, ═NCH 3 It is one of the following: Y 1 From Y 6 each independently represents oxygen, sulfur, selenium, an aryl group, an alkyl group, an alkoxy group, an aryloxy group, -BH 2 , -NH, -NCH 3 , -N(C(=O)CH 3 ) is any one of Q 1 , Q 2 are either oxygen, sulfur, or methylene groups. A capped polynucleotide, characterized in that:

2. E 1 From E 5 Each of Z is a carbon; 1 From Z 5 each independently is carbon or oxygen; 1 and Z 1 Between E 2 and Z 2 Between E 3 and Z 3 Between E 4 and Z 4 Between E 5 and Z 5 and at least one carbon-carbon double bond is present between B 1 From B 4 each of which is independently a nucleobase, m 1 , m 2 are each 0 or 1, n 1 is 1, and n 1 and 2 and 3 The sum of is 5 or less, G 1 , G 2 are each hydrogen or a methyl group, R 1 From R 6 each independently represents one of hydrogen, a hydroxyl group, and an alkoxy group; X 1 From X 6 each independently represents either oxygen or sulfur; Y 1 From Y 6 each independently represents either oxygen or sulfur; Q 1 , Q 2 are either oxygen or sulfur 2. The capped polynucleotide of claim 1.

3. E 1 From E 5 Each of Z is a carbon; 1 From Z 5 each independently is carbon or oxygen; 1 and Z 1 Between E 2 and Z 2 Between E 3 and Z 3 Between E 4 and Z 4 Between E 5 and Z 5 and at least one carbon-carbon double bond is present between B 1 From B 4 each of which is independently a nucleobase, n 1 is 1, and n 1 and 2 and 3 The sum of is 5 or less, m 1 , m 2 are each 0 or 1, G 1 , G 2 are each hydrogen or a methyl group, R 1 From R 6 each independently represents one of a hydroxyl group and an alkoxy group; X 1 From X 6 each independently represents either oxygen or sulfur; Y 1 From Y 6 each independently represents either oxygen or sulfur; Q 1 , Q 2 are either oxygen or sulfur 2. The capped polynucleotide of claim 1.

4. E 1 From E 5 Each of Z is a carbon; 1 From Z 5 each independently is carbon or oxygen; 1 and Z 1 Between E 2 and Z 2 Between E 3 and Z 3 Between E 4 and Z 4 Between E 5 and Z 5 and at least one carbon-carbon double bond is present between B 1 From B 4 each of which is independently a nucleobase, m 1 , m 2 are all 1, n 1 is 1, and n 1 and 2 and 3 The sum of is 5 or less, G 1 , G 2 are each hydrogen or a methyl group, R 1 , R 4 From R 6 each of which is a hydroxyl group, and R 2 and R 3 each is an alkoxy group, or R 2 , R 4 From R 6 each of which is a hydroxyl group, and R 1 and R 3 each of which is an alkoxy group; X 1 From X 6 each independently represents either oxygen or sulfur; Y 1 From Y 6 each independently represents either oxygen or sulfur; Q 1 , Q 2 are either oxygen or sulfur 2. The capped polynucleotide of claim 1.

5. The capped polynucleotide is a compound represented by the following structural formula: B 1 , B 2 are the nucleobases, G 1 , G 2 are each hydrogen or a methyl group, R 1 is a hydroxyl group and R 2 is a methoxy group, or R 2 is a hydroxyl group and R 1 is a methoxy group, X 1 From X 4 each independently represents either oxygen or sulfur; Y 1 From Y 4 each independently represents either oxygen or sulfur; Q 1 , Q 2 are either oxygen or sulfur 2. The capped polynucleotide of claim 1.

6. The capped polynucleotide is a compound represented by the following structural formula: In the above structural formula, E 1 From E 5 Each of Z is a carbon; 1 From Z 5 are independently carbon or oxygen, and at least one pentose is connected to the adjacent phosphate group by a carbon-carbon double bond; B 1 From B 4 each of which is independently a nucleobase, n 1 is 1, and n 1 and 2 and 3 The sum of is 5 or less, R 1 From R 6 each independently represents one of hydrogen, a hydroxyl group, and a methoxy group; X 4 is oxygen or sulfur 2. The capped polynucleotide of claim 1.

7. The carbon-carbon double bond in the structural formula is at least one of E-type and Z-type. A capped polynucleotide according to any one of claims 1 to 6.

8. The capped polynucleotide of any one of claims 1 to 6, wherein the capped polynucleotide is any one of the following compounds or salts:

9. The capped polynucleotide of any one of claims 1 to 6, wherein the capped polynucleotide is any one of the following compounds or salts:

10. The nucleobase includes any one of a natural nucleobase and a modified nucleobase, Optionally, the natural nucleobase is any one selected from adenine, uracil, guanine, hypoxanthine, cytosine, thymine, an adenine derivative, a uracil derivative, a guanine derivative, a cytosine derivative, and a thymine derivative; Optionally, in said modified nucleobase, the modifying group is N 6 -methyladenine, N 1 -methyladenine, N 6 -2'-O-dimethyladenosine, pseudouridine, N 1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5-methyluridine, pseudoisocytosine, 5-methoxycytosine, 2-thiocytosine, 5-hydroxycytosine, N 1 -methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, N 1 -methylguanine, N 1 - any one selected from methylguanine and isoguanine A capped polynucleotide according to any one of claims 1 to 6.

11. The components include a template DNA and a capped polynucleotide according to any one of claims 1 to 10, wherein the template DNA is provided with a transcription position complementary to the capped polynucleotide. A composition for preparing capped mRNA, characterized by:

12. 12. A method for preparing capped mRNA comprising the steps of: performing transcription using the composition for preparing capped mRNA of claim 11. A method for preparing capped mRNA, comprising:

13. The polynucleotide comprises a main component of an mRNA fragment and a polynucleotide to which the cap of any one of claims 1 to 10 has been added, and the polynucleotide to which the cap has been added is bound to the 5' end of the main component of the mRNA fragment. A capped mRNA characterized by:

14. The method includes a step of translating the capped mRNA of claim 13 as a template. A method for preparing a pharmaceutical protein, comprising:

15. It is prepared by the method for preparing a pharmaceutical protein according to claim 14. A pharmaceutical protein characterized by:

16. Use of the capped mRNA of claim 13 in a medicine or vaccine for the treatment of cancer.

17. The method comprises the capped mRNA of claim 13 and a vector carrying the capped mRNA, wherein the vector comprises any one of a lipid nanoparticle, a liposome, a polymer nanoparticle, a solid lipid nanoparticle, and an emulsion. A pharmaceutical preparation characterized by:

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