Deuterated nucleotide and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to deuterated nucleotide shown in a formula (I) and application thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a deuterated nucleotide represented by formula (I) and application thereof. BACKGROUND

[0002] As a new generation of biological medicine platform, mRNA has shown great potential in the fields of vaccine development, protein replacement therapy, tumor immunotherapy, and gene editing therapy. Although mRNA technology has made breakthrough progress in the new crown vaccine, its broader clinical application still faces two key challenges: first, mRNA molecules are easily recognized by the innate immune system, triggering inflammatory responses, which limits the effective dose window; second, the translation efficiency and stability of mRNA still have room for improvement, which directly affects the yield and duration of therapeutic proteins.

[0003] The immunogenicity and translation efficiency of mRNA are regulated by nucleotide composition: on the one hand, unmodified uracil nucleotides are easily recognized and activated by pattern recognition receptors (such as TLR7 / 8, RIG-I, etc.) to activate the type I interferon pathway (Karikó et al., 2005, Immunity); on the other hand, traditional chemical modifications (such as 5-methoxyuridine (5moU)) can reduce immunogenicity, but have limited effect on improving translation efficiency. In the prior art, the COVID-19 vaccines (mRNA-1273 and BNT162b) of Moderna and BioNTech use m1Ψ to replace uridine (U) completely, which significantly reduces the immunogenicity of mRNA (by inhibiting the activation of receptors such as TLR7 / 8 and RIG-I), while improving the translation efficiency (enhancing ribosome binding ability). However, m1Ψ cannot completely eliminate immunogenicity and may still activate innate immune responses through secondary pathways such as TLR3, leading to inflammatory side effects in some patients; m1Ψ also has problems with translation fidelity, which may cause ribosome misreading at high doses, leading to truncated proteins or abnormal translation slippage (Mulroney et al., 2024, Nature).

[0004] Therefore, developing new nucleotide modifications that can effectively avoid innate immune recognition, enhance translation activity, and maintain translation fidelity is the key to breaking the current bottleneck in mRNA drug development. SUMMARY

[0005] The present application provides a deuterated nucleotide, which can be used for in vitro transcription of mRNA, and can reduce innate immunity and improve the expression level and translation fidelity of mRNA coded proteins.

[0006] Nucleotides

[0007] The first aspect of the present application provides a nucleotide or a pharmaceutically acceptable salt thereof, wherein the nucleotide has a structure represented by formula (I)

[0008]

[0009] wherein,

[0010] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 each independently H or D, provided that at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 is D;

[0011] R 12 is selected from

[0012] In some embodiments, at least one of R 5 , R 10 , R 11 is D.

[0013] In some embodiments, R 5 is D.

[0014] In some embodiments, R 5 has a deuterium content of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. In some embodiments, R 5 has a deuterium content of at least 95%. In some embodiments, R 5 has a deuterium content of at least 98%.

[0015] In some embodiments, R 10 , R 11 is D.

[0016] In some embodiments, R 10 , R 11having a deuterium content of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. In some embodiments, R 10 , R 11 having a deuterium content of at least 95%. In some embodiments, R 10 , R 11 having a deuterium content of at least 98%.

[0017] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 6 , R 7 , R 8 , R 9 is H.

[0018] In some embodiments, R 12 is

[0019] In some embodiments, R 12 is

[0020] In some embodiments, the compound of Formula (I) is selected from a compound of Formula (I-1),

[0021]

[0022] wherein R 5 , R 10 , R 11 , R 12 are as defined in any embodiment of the application.

[0023] In some embodiments, the nucleotide is selected from

[0024]

[0025] In some embodiments, the nucleotide is selected from

[0026]

[0027] Nucleosides

[0028] A second aspect of the application provides a nucleoside, or a pharmaceutically acceptable salt thereof, wherein the nucleoside has a structure according to Formula (II)

[0029]

[0030] wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 as defined in any embodiment of the present application.

[0031] In some embodiments, the nucleoside is selected from

[0032]

[0033] Polynucleotides

[0034] A third aspect of the present application provides a polynucleotide encoding a polypeptide and / or protein of interest, wherein the polynucleotide comprises one or more modified uracil nucleotides or nucleosides derived from the nucleotide or a pharmaceutically acceptable salt thereof of the first aspect of the present application or the nucleoside or a pharmaceutically acceptable salt thereof of the second aspect of the present application.

[0035] In some embodiments, the modified uracil nucleotide or nucleoside comprises or has a structure represented by Formula (III)

[0036]

[0037] wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 as defined in any embodiment of the present application.

[0038] In some embodiments, the structure represented by Formula (III) is selected from

[0039]

[0040] In some embodiments, the modified uracil nucleotide comprises or has a structure represented by Formula (III-1)

[0041]

[0042] wherein R 1R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 R 10 R 11 as defined in any embodiment of the application.

[0043] In some embodiments, the structure represented by Formula (III-1) is selected from

[0044]

[0045] In some embodiments, each position represented by D in the nucleotide or a pharmaceutically acceptable salt thereof of the first aspect, the nucleoside or a pharmaceutically acceptable salt thereof of the second aspect, the polynucleotide encoding the polypeptide and / or protein of interest of the third aspect of the application has a deuterium content of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%. In some embodiments, each position represented by D has a deuterium content of at least 95%. In some embodiments, each position represented by D has a deuterium content of at least 98%.

[0046] In some embodiments, the uracil nucleotides in the polynucleotide encoding the polypeptide and / or protein of interest are replaced, in whole or in part, by the structure represented by Formula (III) or Formula (III-1).

[0047] In some embodiments, the polynucleotide encoding the polypeptide and / or protein of interest comprises (or is) a sequence of n number of linked nucleotides selected from guanosine, adenosine, cytidine, and the nucleotide or a pharmaceutically acceptable salt thereof of the first aspect of the application. It will be appreciated by a person skilled in the art that each nucleotide is linked by a bond, for example, a phosphodiester bond.

[0048] Nucleic acid molecules

[0049] The fourth aspect of the application provides a nucleic acid molecule comprising the polynucleotide encoding the polypeptide and / or protein of interest of the third aspect of the application.

[0050] In some embodiments, the nucleic acid molecule further comprises one or more of a 5’ cap structure, a 5’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR), a 3’ terminal poly-A tail (3’ poly-A tail).

[0051] In some embodiments, the nucleic acid molecule comprises, in order from 5’ end to 3’ end: a 5’ cap structure, a 5’ UTR, a polynucleotide encoding a polypeptide and / or protein of interest, and a 3’ UTR.

[0052] In some embodiments, the 5’ cap structure is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0053] In some embodiments, the nucleic acid molecule is selected from the group consisting of small interfering RNA (siRNA), messenger RNA (mRNA), short hairpin (shRNA), antisense oligonucleotide, and RNA aptamer.

[0054] In some embodiments, the nucleic acid molecule is mRNA.

[0055] In some embodiments, the nucleic acid molecule is isolated.

[0056] In some embodiments, the nucleic acid molecule is purified.

[0057] Vectors

[0058] A fifth aspect of the present application provides a vector comprising the nucleic acid molecule of the fourth aspect of the present application.

[0059] In some embodiments, the vector is selected from the group consisting of a plasmid, a bacteriophage, and a viral vector.

[0060] Delivery compositions

[0061] A sixth aspect of the present application provides a delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of: the polynucleotide encoding a polypeptide and / or protein of interest of the third aspect of the present application, the nucleic acid molecule of the fourth aspect of the present application, and the vector of the fifth aspect of the present application.

[0062] In some embodiments, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, and a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0063] In some embodiments, the delivery vehicle is a nanolipid particle.

[0064] In some embodiments, the nanolipid particle comprises one or more of a cationic lipid, a pegylated lipid, a neutral lipid, and a steroid or steroid analog.

[0065] In some embodiments, the nanolipid particle comprises ionizable cationic lipid SM-102, helper lipid DSPC, cholesterol, and a PEGylated lipid PEG2000-DMG.

[0066] Pharmaceutical compositions

[0067] A seventh aspect of the present application provides a pharmaceutical composition comprising the polynucleotide encoding a polypeptide and / or protein of the third aspect of the present application, the nucleic acid molecule of the fourth aspect of the present application, the vector of the fifth aspect of the present application, and / or the delivery composition of the sixth aspect of the present application, and one or more pharmaceutically acceptable carriers and / or excipients.

[0068] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0069] In some embodiments, the additional pharmaceutically active agent is provided as a separate component or as a mixed component with the polynucleotide encoding a polypeptide and / or protein, the nucleic acid molecule, the vector, and / or the delivery composition.

[0070] The pharmaceutical composition of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the pharmaceutical composition of the present application in the required amount in an appropriate solvent with one or more of the other ingredients, including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof, and filtering sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for ease of storage and use. Such lyophilized powders can be reconstituted into sterile injection solutions just prior to use. The pharmaceutical composition of the present application can also be formulated as a sterile, lyophilized powder for reconstitution with a suitable solvent, such as sterile water or normal saline, just prior to use.

[0071] In addition, the polynucleotide encoding a polypeptide and / or protein of the present application, the nucleic acid molecule, the vector, or the delivery composition can be present in the pharmaceutical composition in unit dosage form to facilitate administration.

[0072] The pharmaceutical compositions of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracerebroventricular, inguinal, intravesical, local (e.g., powder, salve or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, intratumoral injection). It will be understood by those skilled in the art that the route and / or means of administration will vary depending on the intended purpose. In some preferred embodiments, the pharmaceutical compositions of the present application are administered by intravenous infusion or injection.

[0073] The pharmaceutical compositions of the present application can include a therapeutically effective amount or a prophylactically effective amount of a polynucleotide, nucleic acid molecule, vector or delivery composition encoding a polypeptide and / or protein of interest. The effective amount of a polynucleotide, nucleic acid molecule, vector or delivery composition encoding a polypeptide and / or protein of interest can vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, body weight, and gender, the mode of administration of the drug, and other therapies being administered concurrently, and the like.

[0074] In the present application, the dosing regimen can be adjusted to achieve the best response for the intended purpose (e.g., therapeutic or prophylactic response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dose can be proportionally reduced or increased depending on the urgency of the therapeutic situation.

[0075] Kits

[0076] The eighth aspect of the present application provides a kit comprising the nucleotide of the first aspect of the present application or a pharmaceutically acceptable salt thereof.

[0077] In some embodiments, the kit further comprises adenosine triphosphate (ATP), guanosine triphosphate (GTP), and cytidine triphosphate (CTP).

[0078] Uses

[0079] The ninth aspect of the present application provides use of the nucleoside of the second aspect of the present application or a pharmaceutically acceptable salt thereof in the manufacture of the nucleotide of the first aspect of the present application or a pharmaceutically acceptable salt thereof.

[0080] The tenth aspect of the present application provides use of the nucleotide or a pharmaceutically acceptable salt thereof of the first aspect of the present application, or the nucleoside or a pharmaceutically acceptable salt thereof of the second aspect of the present application, or the kit of the eighth aspect of the present application in the manufacture of the polynucleotide encoding the polypeptide and / or protein of interest of the third aspect of the present application, the nucleic acid molecule of the fourth aspect of the present application, the vector of the fifth aspect of the present application, the delivery composition of the sixth aspect of the present application, or the pharmaceutical composition of the seventh aspect of the present application.

[0081] The eleventh aspect of the present application provides use of the polynucleotide encoding the polypeptide and / or protein of interest of the third aspect of the present application, the nucleic acid molecule of the fourth aspect of the present application, the vector of the fifth aspect of the present application, the delivery composition of the sixth aspect of the present application, or the pharmaceutical composition of the seventh aspect of the present application in the manufacture of a medicament for treating and / or preventing a disease or disorder or alleviating the severity of the disease or disorder.

[0082] The twelfth aspect of the present application provides the polynucleotide encoding the polypeptide and / or protein of interest of the third aspect of the present application, the nucleic acid molecule of the fourth aspect of the present application, the vector of the fifth aspect of the present application, the delivery composition of the sixth aspect of the present application, or the pharmaceutical composition of the seventh aspect of the present application for use in treating and / or preventing a disease or disorder or alleviating the severity of the disease or disorder.

[0083] The thirteenth aspect of the present application provides a method of treating and / or preventing a disease or disorder or alleviating the severity of the disease or disorder, comprising administering to an individual an effective amount of the polynucleotide encoding the polypeptide and / or protein of interest of the third aspect of the present application, the nucleic acid molecule of the fourth aspect of the present application, the vector of the fifth aspect of the present application, the delivery composition of the sixth aspect of the present application, or the pharmaceutical composition of the seventh aspect of the present application.

[0084] In some embodiments, the individual is administered a single dose, two doses, three doses or more doses of the polynucleotide, nucleic acid molecule, vector, delivery composition or pharmaceutical composition and optionally an additional dose of the polynucleotide, nucleic acid molecule, vector, delivery composition or pharmaceutical composition.

[0085] In some embodiments, the individual is a mammal, for example a human.

[0086] Definitions of terms

[0087] In the present application, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, for better understanding of the present application, the definitions and explanations of relevant terms are provided below.

[0088] The terms "a" and "an," and the like, as used in describing the present application (especially in the context of the following claims), should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0089] As used herein, the term "and / or", shall, wherever following a term or term group that two or more specified things are to be taken together, be taken to mean one of either the specified things by itself or any combination of the specified things. Thus, the term "and / or" as used in a phrase such as "A and / or B" is intended to encompass "A and B," "A or B," "A" (alone), and "B" (alone).

[0090] Unless otherwise indicated, when a position is designated as "H" or "hydrogen", or its chemical representation implies hydrogen, it is to be understood as hydrogen having natural abundance isotopic composition.

[0091] Unless otherwise indicated, when a position is designated as "D" or "deuterium", it is to mean that the deuterium content (incorporation, abundance or enrichment) is at least 50%. In some embodiments, the deuterium content (incorporation, abundance or enrichment) is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. The deuterium content can be measured by techniques known in the art, such as H NMR spectroscopy. 1

[0092] As used herein, the term "deuterated" refers to a compound or group in which one or more hydrogens are replaced by deuterium.

[0093] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present application that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include acid addition salts, formed with inorganic acids or with organic acids, or coordinate compounds formed with organic bases.

[0094] ​Pharmaceutically acceptable salts of the compounds of this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acid, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acid, or by using other methods such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0095] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and

[0096] Pharmaceutically acceptable salts also include, without limitation, those derived from the following acids: 2,2-dichloroacetic, acetic, adipic, ascorbic, aspartic, benzenesulfonic, bis- methylnaphtalenesulfonic, boric, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrochloric, maleic, malic, malonic, mandelic, methanesulfonic, naphthalenesulfonic, nitric, nitrous, oxalic, pamoic, pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric, toluenesulfonic, and xinafoic.

[0097] As used herein, the term "nucleic acid" refers to a polymer comprising or consisting of nucleotide monomers covalently linked to one another by phosphodiester bonds. Nucleic acids can also encompass modified nucleic acid molecules, such as DNA or RNA molecules with base modifications, sugar modifications, or backbone modifications, e.g., deuterium modifications. Nucleic acids can exist in various forms, such as: isolated segments of a sequence and recombinant vectors or recombinant polynucleotides encoding a polypeptide, such as one or both chains of an antigen or antibody, or fragments, derivatives, muteins, or variants thereof; polynucleotides sufficient for use as a hybridization probe; PCR primers or sequencing primers for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide.

[0098] As used herein, the term "polynucleotide" refers to a nucleic acid molecule, which can be recombinant or isolated from total genomic nucleic acid. In certain embodiments, a polynucleotide includes regulatory sequences that are substantially isolated from their naturally occurring gene or protein-encoding sequences. A polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA or synthetic), an analog thereof, or a combination thereof. Additional coding or non-coding sequences can or can not be present within the polynucleotide.

[0099] As used herein, the term "RNA" means a nucleic acid molecule comprising nucleotides such as adenosine monophosphate, uridine monophosphate, guanosine monophosphate, cytidine monophosphate, and modified uridine monophosphate monomers, connected to one another along a so-called backbone. The backbone is formed by a phosphodiester bond between the sugar (e.g., ribose) of a first monomer and the phosphate moiety of a second adjacent monomer. RNA can be obtained, for example, within a cell from transcription of a DNA sequence. In eukaryotic cells, transcription is typically carried out within the nucleus or the mitochondria. In vivo, transcription of DNA can produce immature RNA, which is processed into messenger RNA (mRNA). Processing of immature RNA, for example, in eukaryotic organisms, includes various post-transcriptional modifications such as splicing, 5' capping, polyadenylation, export from the nucleus or mitochondria. The nucleotide sequence is processed into mature messenger RNA and provides an amino acid sequence that can be translated into a peptide or protein. The mature mRNA can include a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a poly-A tail sequence. RNA can comprise all or mostly ribonucleotide residues. In one embodiment, RNA can be messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As known to those skilled in the art, mRNA generally contains a 5' untranslated region (5' UTR), a polypeptide coding region, and a 3' untranslated region (3' UTR). Without any limitation, RNA can encompass double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinantly produced RNA, and modified RNA.

[0100] As used herein, the terms "protein," "polypeptide" are used synonymously herein and refer to a polymer of amino acid monomers, e.g., a molecule comprising at least two amino acid residues. A polypeptide can include a gene product, a naturally occurring polypeptide, a synthetic polypeptide, a homolog, a xenolog, an isolog, a fragment, and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or can be a multimeric complex, such as a dimer, a trimer, or a tetramer. A protein comprises one or more peptides or polypeptides and can fold into a 3-dimensional form that can be required for the protein to perform its biological function.

[0101] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, in pharmacological and / or physiological terms, with the subject and active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable carriers and / or excipients include, but are not limited to, pH adjusting agents, surfactants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to retard absorption, preservatives, stabilizers. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to retard absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the pharmaceutical, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried lactose, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), and the like.

[0102] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention means an amount sufficient to prevent, stop, or delay the onset of a disease; an effective amount for disease treatment means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0103] As used in this application, the term "treatment" aims to alleviate, reduce, improve, or eliminate a targeted disease state or symptom. A subject is successfully "treated" if one or more indicators and symptoms of a subject show an observable and / or detectable reduction or improvement. It should also be understood that treatment of a disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0104] As used in this application, the term "prevention" aims to avoid, reduce, prevent, or delay the onset of a disease or disease-related symptoms before the onset of the relevant drug. "Prevention" does not necessarily require the complete prevention of the onset of a disease or disease-related symptoms. For example, reducing the risk of a subject developing a specific disease or disease-related symptoms after the administration of the relevant drug, or lessening the severity of subsequently occurring related symptoms, can be considered as "prevention" of the onset or development of the disease.

[0105] Beneficial effects

[0106] The deuterated nucleotides provided by this invention can increase the level of proteins expressed by mRNA, reduce innate immune activation, and increase translational fidelity, making the optimized mRNA molecules have broad application prospects in the fields of vaccines, gene therapy, and antibody therapy. Attached Figure Description

[0107] Figure 1 Electrophoresis diagram of chemically modified mRNA (1).

[0108] Figure 2 . Results of toxicity testing of chemically modified mRNA in cells.

[0109] Figure 3 Parameter detection results of chemically modified mRNA lipid nanoparticles.

[0110] Figure 4 The expression results of chemically modified mRNA in mice.

[0111] Figure 5 Innate immune response levels of chemically modified mRNA in mice.

[0112] Figure 6 Electropherogram of chemically modified mRNA (2).

[0113] Figure 7 Detection results of translation frameshift rate of chemically modified mRNA.

[0114] Wherein, * in the figure represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

[0115] Sequence information

[0116] The information of part of the sequences involved in the present application is shown in Table 1 below.

[0117] Table 1. Information of part of the sequences

[0118]

[0119]

[0120]

[0121]

[0122] DETAILED DESCRIPTION

[0123] Embodiments of the present application will be described in detail below with reference to Examples, but those skilled in the art will understand that the following Examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the Examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not noted for the manufacturer are all conventional products that can be obtained by purchase on the market.

[0124] Example 1: Preparation of ((2R,3S,4R,5R)-3,4-dihydroxy-5-(5-methoxy-2,4-dioxo-3,4- dihydro-pyrimidin-1(2H)-yl-6-d)tetrahydrofuran-2-yl)methyl tetrahydro-pyrophosphonate (2583U)

[0125]

[0126] Step 1) 5-methoxy-pyrimidine-2,4(1H,3H)-dione-6-d

[0127] Sodium metal (138 mg, 6.0 mmol) was added portionwise to deuterium oxide (2.0 mL) under ice bath, stirred for 30 minutes. Then 5-methoxyuracil (265 mg, 1.87 mmol) was added, the system was replaced by argon, and the mixture was heated to 125 °C in a sealed tube for 16 hours. After the reaction was cooled to room temperature, it was acidified with 1.0 N hydrochloric acid, filtered, and the filter cake was dried to give the title product as a white solid 240 mg, yield: 90%. LC-MS (ESI): m / z = 166.1 [M+Na + ].

[0128] Step 2) (2R,3S,4R,5R)-2-((benzoyloxy)methyl)-5-(5-methoxy-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl-6-d)tetrahydrofuran-3,4-dicarboxylate

[0129] To a solution of 5-methoxyuracil-2,4(lH,3H)-dione-6-d (240 mg, 1.68 mmol) in anhydrous acetonitrile (20 mL) was added N,O-bistrimethylsilylacetamide (BSA) (1.25 mL, 5.04 mmol), and the resulting mixture was heated to reflux for 1 hour. After cooling to room temperature, l-acetyl-tri-benzyloxy-ribofuranose (1.1 g, 2.18 mmol) and trimethylsilyl trifluoromethanesulfonate (0.40 mL, 2.18 mol) were added. The resulting reaction mixture was heated to reflux for 2 hours. After the reaction was cooled to room temperature, it was rotary evaporated under reduced pressure, and the resulting residue was purified by column chromatography (petroleum ether: ethyl acetate = 1: 1) to give the title product as a white solid 600 mg, yield: 61%. LC-MS (ESI): m / z = 588.1 [M+H + ].

[0130] Step 3) l-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5- methoxyuracil-2,4(lH,3H)-dione-6-d

[0131] (2R,3S,4R,5R)-2-((benzoyloxy)methyl)-5-(5-methoxy-2,4-dioxo-3,4- dihydropyrimidin-l(2H)-yl-6-d)tetrahydrofuran-3,4-dicarboxylate (370 mg, 0.63 mmol) was added to a 7 N solution of ammonia in methanol (6 mL), and the mixture was heated to 45 °C for 24 hours. After rotary evaporation under reduced pressure, the resulting residue was slurried with ethyl acetate, acetonitrile, and then purified by column chromatography to give the title product as a white solid 145 mg, yield: 83%. LC-MS (ESI): m / z = 276.0 [M+H + ]. 1H-NMR (500 MHz, CD3OD) δ 5.98-5.95 (m, 1H), 4.24-4.21 (m, 2H), 4.06-4.03 (m, 1H), 3.93-3.89 (m, 1H), 3.81-3.77 (m, 1H), 3.72 (s, 3H).

[0132] Step 4: ((2R,3S,4R,5R)-3,4-dihydroxy-5-(5-methoxy-2,4,-dioxo-3,4-dihydropyrimidin- 1 (2H)-yl-6-d)tetrahydrofuran-2-yl)methyl tetrahydrogen pyrophosphoric acid (2583U)

[0133] To a solution of l-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2- yl)-5-methoxypyrimidine-2,4(lH,3H)-dione-6-d (50 mg, 0.182 mmol) in trimethyl phosphate (2 mL) was added phosphorous oxychloride (25 μL, 0.273 mmol) dropwise at 0 °C under argon. The resulting mixture was stirred for half an hour. Additional phosphorous oxychloride (25 μL, 0.273 mmol) was added and stirred for 1 hour. Pyrophosphoric acid (96 mg, 0.545 mmol) and tripropylamine (0.4 mL, 2.182 mmol) in acetonitrile (2 mL) was added and stirred at room temperature for 2 hours. Diluted with water to 30 mL and purified with 650Q ion column (eluent: 1 M triethylammonium bicarbonate buffer). The product tube was collected and concentrated. The product was purified with reverse phase column three times. The product was purified with 650Q ion column (eluent: 1 M triethylammonium bicarbonate buffer) to give the title product as triethylamine salt 60 mg, yield: 32%. Deuterium incorporation: 98%. 1 H NMR (500 MHz, D2O) δ 6.07 (s, 1H), 4.47 (s, 2H), 4.32-4.28 (m, 2H), 4.24-4.22 (m, 1H), 3.83 (s, 3H). 31 P NMR (202 MHz, D2O) δ -10.80 (d, J = 19.6 Hz, IP), -11.74 (d, J = 19.9 Hz, IP), -22.30 (t, J = 19.3 Hz, IP).

[0134] Example Two: Preparation of ((2R,3S,4R,5R)-3,4-dihydroxy-5-(5-methoxy-2,4,-dioxo- 3,4-dihydropyrimidin-l(2H)-yl)tetrahydrofuran-2-yl)methyl-d2 tetrahydrogen pyrophosphoric acid (2586U)

[0135]

[0136] Step 1) 1-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4- d] [ 1,3 ]dioxol-4-yl)-5-methoxy pyrimidine-2,4( 1 H,3H)-dione

[0137] To a solution of 5-methoxyuridine (2.4 g, 8.75 mmol) in acetone (35 mL) was added p-toluenesulfonic acid monohydrate (1.58 g, 8.31 mmol) and 2,2-dimethoxypropane (5.3 mL, 43.7 mmol). The resulting mixture was stirred at room temperature for 2 hours. Sodium bicarbonate (768 mg, 9.14 mmol) was added and after stirring for 10 minutes, it was concentrated and purified by column chromatography (dichloromethane:methanol = 50:1 to 25:1) to give the title product as a solid 2.52 g, yield: 92%. LC-MS (ESI): m / z = 314.9 [M+H] + .

[0138] Step 2) tert-Butyl (3aS,4S,6R,6aR)-6-(5-methoxy-2,4-dioxo-3,4-dihydropyrimidin- 1 (2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxole-4-carboxylate

[0139] To a solution of 1-((3aR,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuro[3,4- d] [ 1,3 ]dioxol-4-yl)-5-methoxy pyrimidine-2,4( 1 H,3H)-dione (2.33 g, 7.41 mmol) in dichloromethane (50 mL) and tert-butanol (14 mL) was added PDC (4.4 g, 14.8 mmol) and acetic anhydride (6.95 mL, 74.1 mmol). The resulting mixture was stirred at room temperature for 2.5 hours. It was concentrated and purified by column chromatography (petroleum ether: ethyl acetate = 2:1 to 1 :1) to give the title product as a solid 1.8 g, yield: 63%. LC-MS (ESI): m / z = 384.9 [M+H] + .

[0140] Step 3) 1-((3aR,4R,6R,6aR)-6-(hydroxymethyl-d2)-2,2-dimethyltetrahydrofuro[3,4- d] [ 1,3 ]dioxol-4-yl)-5-methoxy pyrimidine-2,4( 1 H,3H)-dione

[0141] To a solution of tert-butyl (3aS,4S,6R,6aR)-6-(5-methoxy-2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxane-4- carboxylate (930 mg, 2.42 mmol) in tetrahydrofuran (10 mL), deuterated methanol (2 mL) and heavy water (1 mL) was added sodium borodeuteride (304 mg, 7.26 mmol) portionwise under ice bath. After the addition, the reaction was warmed to 50 °C for 4 h. The pH was adjusted to about 7 with deuterated acetic acid, water was added, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:1 to 0:1) to give the title product as a white solid 420 mg, yield: 55%. LC-MS (ESI): m / z = 317.0 [M+H] + .

[0142] Step 4) 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl-d2)tetrahydrofuran-2-yl)-5- methoxy pyrimidine-2,4(1H,3H)-dione

[0143] To a solution of 1-((3aR,4R,6R,6aR)-6-(hydroxymethyl-d2)-2,2-dimethyltetrahydrofuro[3,4- d][1,3]dioxol-4-yl)-5-methoxypyrimidine-2,4(1H,3H)-dione (420 mg, 1.33 mmol) in trifluoroacetic acid (3 mL) and water (3 mL) was stirred at room temperature for 2 h. The mixture was concentrated, dissolved in ethanol and concentrated again. The residue was slurried with ethyl acetate to give the title product as a white solid 349 mg, yield: 95%. LC-MS (ESI): m / z = 276.9 [M+H] + .

[0144] Step 5) ((2R,3S,4R,5R)-3,4-dihydroxy-5-(5-methoxy-2,4,-dioxo-3,4-dihydropyrimidin-1(2H)- yl)tetrahydrofuran-2-yl)methyl-d2 tetrahydrogen triphosphate (2586U)

[0145] Referring to Example 1, the title product was obtained by using 1-((2R,3R,4S,5R)-3,4- dihydroxy-5-(hydroxymethyl-d2)tetrahydrofuran-2-yl)-5-methoxypyrimidine-2,4(1H,3H)- dione instead of 1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)- 5-methoxypyrimidine-2,4(1H,3H)-dione-6-d as 40 mg, yield: 27%. Deuterium enrichment: 98.5%. MS (ESI): m / z = 515.0 [M-H]- . 1 H NMR (500 MHz, D20) δ 7.41 (s, 1H), 6.08 (d, J = 5.5 Hz, 1H), 4.51-4.48 (m, 2H), 4.34 (s, 1H), 3.85 (s, 3H). 31 P NMR (202 MHz, D20) δ -10.21 (d, J = 19.1 Hz, 1P), -11.67 (d, J = 20.0 Hz, 1P), -23.18 (t, J = 19.9 Hz, 1P).

[0146] Example Three: In vitro transcription (IVT)

[0147] PCR method was used to prepare linearized template for IVT, using Novagen 2x Phanta Flash Master Mix. PCR reaction was performed in the following system, reaction condition was 98 °C for 5 seconds, 98 °C for 10 seconds, 58 °C for 5 seconds, 72 °C for 10 seconds for 25 cycles, then 72 °C for 1 minute, then 4 °C stop.

[0148] Table 2 PCR reaction system (50 μΐ)

[0149]

[0150] PCR amplification was performed using EGFP (SEQ ID NO: 1) and Fluc (Firefly Luciferase) (SEQ ID NO: 2) as plasmid template, respectively. The primer sequences for amplifying the plasmid template were as follows:

[0151] HBB-F: CGTTGTAAAACGACGGCCAGAGAATTC (SEQ ID NO: 5);

[0152] HBB-R:

[0153] TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAATGAAAATAAATGTTTTTTATTAGG (SEQ ID NO: 6).

[0154] Promega (Cat No: A9282) kit was used to recover IVT template (SEQ ID NO: 1 and SEQ ID NO: 2) in PCR product.

[0155] IVT system was configured according to the following system configuration using natural nucleotides and modified nucleotides to transcribe mRNA:

[0156] Table 3 IVT reaction system (20 μL)

[0157]

[0158]

[0159] Among them, the NTPs of the control group are composed of natural nucleotides ATP, GTP, CTP and UTP, the NTPs of experimental group 1 are composed of natural nucleotides ATP, GTP, CTP and modified nucleotide 2583U, and the NTPs of experimental group 2 are composed of natural nucleotides ATP, GTP, CTP and modified nucleotide 2586U. IVT reaction was carried out with EGFP (SEQ ID NO: 1) and Fluc (SEQ ID NO: 2) as templates, and the specific operation was as follows.

[0160] After adding the template, incubate at 37°C for 2h, add 1 μL DNase I to each reaction and incubate at 37°C for 30min to digest the DNA template, and precipitate the RNA at -80°C using LiCl precipitation method. Centrifuge to collect the precipitated mRNA, centrifuge at 14,000 rpm for 10 min at 4°C, and white RNA precipitate appears at the bottom of the centrifuge tube; after discarding the supernatant, add 1 mL of pre-cooled 70% ethanol, vortex to wash the RNA, and repeat the washing once; dry at room temperature until the precipitate becomes translucent, add RNase-free water to dissolve the RNA, and measure the mRNA concentration with NanoDrop One, and detect the mRNA quality by 1% TBE agarose gel electrophoresis. The results are shown in Figure 1 It is shown that the mRNA products of EGFP (SEQ ID NO: 1) and Fluc (SEQ ID NO: 2) of higher quality were prepared by replacing natural UTP with modified nucleotides 2583U and 2586U, respectively.

[0161] Example Four: Toxicity detection of chemically modified mRNA in cells

[0162] The toxicity of chemically modified mRNA was detected in A549 cells. DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was used, and 0.3 x 10 6A549 cells were seeded in 96-well plates at a concentration of 1 x 104cells / mL, 100 μL per well. After 12 hours of culture, lipo8000 was used to transfect mRNA into A549 cell lines at a concentration of 1.6:1 ug / mL, wherein the mRNA was EGFP mRNA prepared in Example 3. A control group without any treatment, a group with only 0.16 μL / well of lipo8000 added, and a poly(I:C) group using lipo8000 and poly(I:C) at a concentration of 1.6:1 ug / mL were set up. The transfected cells were incubated in a 37°C cell incubator for 48 h, and the Cell Counting Kit-8 was used to detect the cells using a microplate reader. The results are shown in Figure 2 Chemically modified mRNA is not toxic to A549 cells.

[0163] Example Five: Expression of chemically modified mRNA in mice in vivo

[0164] SM102, DSPC, cholesterol and DMG-PEG2000 were mixed in a molar ratio of 50:10:38.5:1.5 in ethanol, and then the lipid mixture was mixed with Fluc mRNA prepared in Example 3 (dissolved in 50 mM sodium acetate pH 4.0) at a volume ratio of 1:3 using a microfluidic homogenizer, with a total flow rate of 12 mL / min and a flow rate ratio of 1:3. The mRNA-LNP was dialyzed against PBS for 4 h to remove acetic acid and ethanol, and the PBS was replaced every 2 h until the dialysis was neutral, to obtain Fluc-mRNA-LNP. The particle size and distribution of the Fluc-mRNA-LNP were detected using a nanoparticle size analyzer (Malvern), and the encapsulation efficiency of the mRNA was detected using the Ribogreen method (ThermoFisher). The results are shown in Figure 3 The average particle size of the encapsulated vaccine was 60-80 nm, the particle size distribution was uniform (PDI < 0.1), the encapsulation rate was greater than 90%, and the quality was good.

[0165] BALB / c female 6-week-old mice were randomly divided into groups of 5, and injected intramuscularly with Fluc-mRNA-LNP at a dose of 5 μg per mouse. Six hours later, 100 uL of luciferase (Perkin Elmer) substrate was injected intraperitoneally, and live imaging was performed using the AniView100Pro multi-mode animal live imaging system. The results are shown in Figure 4 In BALB / c mice, the expression of Fluc mRNA with modified nucleotide 2586U did not change significantly compared to UTP, while the expression of Fluc mRNA with modified nucleotide 2583U was significantly reduced.

[0166] Example Six: Innate immune response of chemically modified mRNA in mice in vivo

[0167] BALB / c female 6-week-old mice were randomly grouped in 5 mice per group, and injected intramuscularly with Fluc-mRNA-LNP prepared in Example Five at a dose of 5 pg per mouse, and a negative control group was set up to be injected intramuscularly with PBS at a dose of 100 pL per mouse, and a positive control group was injected intramuscularly with poly(I:C) at a dose of 5 pg per mouse, and the spleen was collected 6 hours later, RNA was extracted using RNAiso Plus, reverse transcribed using HyperScript III RT SuperMix for qPCR with gDNA Remover, 2x S6 Universal SYBR qPCR Mix and QuantStudio 7 Flex to quantify the mRNA level of IFNb. The results are shown in Figure 6, in BALB / c mice, compared with UTP, the Fluc mRNA modified with nucleotide 2586U significantly reduced the level of IFNb mRNA stimulated to produce, and the Fluc mRNA modified with nucleotide 2583U had no significant difference in the level of IFNb mRNA stimulated to produce. TM 7Flex quantified the mRNA level of IFNb. The results are shown in Figure 6, in BALB / c mice, compared with UTP, the Fluc mRNA modified with nucleotide 2586U significantly reduced the level of IFNb mRNA stimulated to produce, and the Fluc mRNA modified with nucleotide 2583U had no significant difference in the level of IFNb mRNA stimulated to produce. Figure 5

[0168]

[0169] Example Seven: Detection of translation frame shift rate of chemically modified mRNA

[0170] In order to detect the effect of modified nucleotides on translation fidelity, a CTP was inserted after nucleotides 681-684 (TTTT) of Fluc-WT-2 (SEQ ID NO: 3) to obtain a reporter gene Fluc-ISF-2 (SEQ ID NO: 4) for detecting translation frame shift. Fluc-ISF-2 can only express functional Fluc when the ribosome Frame Shifts over the subsequent CTP at four consecutive UTPs; the expression ratio of Fluc-ISF-2 to Fluc-WT-2 represents the frequency of Frame Shift. The mRNA of Fluc-WT-2 and Fluc-ISF-2 was prepared according to the method of Example Three using the amplification primer sequences (SEQ ID NO: 11) and (SEQ ID NO: 12), and the results are shown in Figure 7. Figure 6

[0171] HBB-F’: GAATTCGCCGTAATACGACTCAC (SEQ ID NO: 11);

[0172] ​​HBB-R': TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCGAGAGCGTAATCTGGAAC (SEQ ID NO: 12).

[0173] The chemically modified Fluc-WT-2 and Fluc-ISF-2 mRNA were transfected into HeLa cells by lipo8000. After 24 hours, the expression of Fluc-WT-2 and Fluc-ISF-2 mRNA was quantified by using the Firefly Luciferase Reporter Gene Detection Kit (Nanjing Keygen Biotech Co., Ltd.) and Perkin Elmer Multifunctional Enzyme Labeling Instrument. The results are shown in Figure 6. In HeLa cells, the mRNA prepared by modifying nucleotide 2586U has a significantly lower frameshift rate than the mRNA prepared by modifying nucleotide 2583U. Figure 7

[0174] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the specific embodiments of the present application can be modified or some technical features can be replaced by equivalent ones; without departing from the spirit of the technical solutions of the present application, they should be covered in the technical solution range of the present application.​

Claims

1. A nucleotide or a pharmaceutically acceptable salt thereof, wherein, The nucleotide has a structure represented by Formula (I) wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 each independently H or D, provided that at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 is D; R 12 selected from 2. The nucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein, R 5 , R 10 , R 11 at least one of R is D; Preferably, R 5 is D; Preferably, R 10 , R 11 is D.

3. The nucleotide or a pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, The nucleotide is selected from 4. The nucleotide or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein, each position represented by D has a deuterium content of at least 70%; preferably, each position represented by D has a deuterium content of at least 95%; preferably, each position represented by D has a deuterium content of at least 98%.

5. A nucleoside or a pharmaceutically acceptable salt thereof, wherein, The nucleoside has a structure represented by Formula (II) wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 as defined in any one of claims 1 to 4.

6. The nucleoside of claim 5, or a pharmaceutically acceptable salt thereof, wherein, The nucleoside is selected from 7. The nucleoside of claim 5 or 6, or a pharmaceutically acceptable salt thereof, wherein, each position represented by D has a deuterium content of at least 70%; preferably, each position represented by D has a deuterium content of at least 95%; preferably, each position represented by D has a deuterium content of at least 98%.

8. Use of the nucleoside or a pharmaceutically acceptable salt thereof of any one of claims 5-7 in the manufacture of the nucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1-4.

9. A polynucleotide encoding a polypeptide and / or protein of interest, wherein, The polynucleotide comprises one or more modified uracil nucleotides or nucleosides derived from the nucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1-4 or the nucleoside or a pharmaceutically acceptable salt thereof of any one of claims 5-7.

10. The polynucleotide encoding a polypeptide and / or protein of interest of claim 9, wherein the modified uracil nucleotide or nucleoside comprises or has a structure represented by Formula (III) wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 as defined in any one of claims 1 to 4; preferably, the structure represented by Formula (III) is selected from preferably, each position represented by D has a deuterium content of at least 70%; preferably, each position represented by D has a deuterium content of at least 95%; preferably, each position represented by D has a deuterium content of at least 98%.

11. A nucleic acid molecule comprising the polynucleotide encoding a polypeptide and / or protein of interest of claim 9 or 10; preferably, the nucleic acid molecule further comprises one or more of a 5’ cap structure, a 5’ UTR, a 3’ UTR, a 3’ poly-A tail; preferably, the nucleic acid molecule comprises, in order from 5’ end to 3’ end: a 5’ cap structure, a 5’ UTR, the polynucleotide encoding a polypeptide and / or protein of interest, a 3’ UTR.

12. The nucleic acid molecule of claim 11, wherein, The nucleic acid molecule is selected from siRNA, mRNA, shRNA, antisense oligonucleotide, and RNA aptamer; preferably, the nucleic acid molecule is mRNA.

13. A vector comprising the nucleic acid molecule of claim 11 or 12. preferably, the vector is selected from plasmid, bacteriophage, and viral vector.

14. A delivery composition comprising a delivery vehicle, and one or more selected from the polynucleotide encoding a polypeptide and / or protein of interest of claim 9 or 10, the nucleic acid molecule of claim 11 or 12, the vector of claim 13. preferably, the delivery vehicle is selected from lipid particle, sugar particle, metal particle, protein particle, liposome, exosome, microvesicle, gene gun, and viral vector (e.g., replication-defective retrovirus, lentivirus, adenovirus, or adeno-associated virus); preferably, the delivery vehicle is nanolipid particle; preferably, the nanolipid particle comprises one or more of cationic lipid, PEGylated lipid, neutral lipid, and steroid or steroid analogue. Preferably, the nano-lipid particle comprises ionizable cationic lipid SM-102, helper lipid DSPC, cholesterol, and a PEGylated lipid PEG2000-DMG.

15. A pharmaceutical composition comprising the polynucleotide encoding a polypeptide and / or protein of interest of claim 9 or 10, the nucleic acid molecule of claim 11 or 12, the vector of claim 13, and / or the delivery composition of claim 14, and one or more pharmaceutically acceptable carriers and / or excipients; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent. Preferably, the additional pharmaceutically active agent is provided as a separate component or as a mixed component with the polynucleotide encoding a polypeptide and / or protein of interest, the nucleic acid molecule, the vector, and / or the delivery composition.

16. A kit comprising the nucleotide of any one of claims 1-4, or a pharmaceutically acceptable salt thereof; Preferably, the kit further comprises adenosine triphosphate (ATP), guanosine triphosphate (GTP), and cytidine triphosphate (CTP).

17. Use of the nucleotide of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or the nucleoside of any one of claims 5-7, or a pharmaceutically acceptable salt thereof, or the kit of claim 16, in the manufacture of the polynucleotide encoding a polypeptide and / or protein of interest of claim 9 or 10, the nucleic acid molecule of claim 11 or 12, the vector of claim 13, the delivery composition of claim 14, or the pharmaceutical composition of claim 15.

18. Use of the polynucleotide encoding a polypeptide and / or protein of interest of claim 9 or 10, the nucleic acid molecule of claim 11 or 12, the vector of claim 13, the delivery composition of claim 14, or the pharmaceutical composition of claim 15, in the manufacture of a medicament for the treatment and / or prevention of a disease or disorder, or for reducing the severity of a disease or disorder.

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