CN120988041A

CN120988041A - Deuterated nucleotides and their applications

Info

Publication number: CN120988041A
Application number: CN202511082389.3A
Authority: CN
Inventors: 张琼; 全韵; 潘喻; 张健存; 邹晴安
Original assignee: Guangzhou National Laboratory
Current assignee: Guangzhou National Laboratory
Priority date: 2025-08-01
Filing date: 2025-08-01
Publication date: 2025-11-21

Abstract

This invention belongs to the field of biomedicine, specifically relating to the deuterated nucleotides shown in formula (I) and their applications.

Description

Deuterated nucleotide and application thereof

Technical Field

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

Background

MRNA as a new generation biological medicine platform has great potential in the fields of vaccine development, protein substitution therapy, tumor immunotherapy, gene editing therapy and the like. Although mRNA technology has made breakthrough progress in new crown vaccines, its broader clinical application still faces two key challenges, namely that mRNA molecules are easily recognized by the innate immune system to trigger inflammatory reactions, resulting in limited effective dose window, and that translation efficiency and stability of mRNA still have room for improvement, directly affecting therapeutic protein yield and duration.

The immunogenicity and translational efficiency of mRNA are regulated by nucleotide composition, on the one hand, unmodified uracil nucleotides are easily recognized by pattern recognition receptors (e.g., TLR7/8, RIG-I, etc.) to activate the type I interferon pathway (Karik d et al 2005, immunity), and on the other hand, traditional chemical modifications (e.g., 5-methoxyuridine (5 moU)) can reduce immunogenicity, but have limited improvement in translational efficiency. In the prior art, the COVID-19 vaccine (mRNA-1273 and BNT162 b) of Moderna and BioNTech adopts m1 ψ to completely replace uridine (U), so that the immunogenicity of mRNA is obviously reduced (by inhibiting receptor activation such as TLR7/8 and RIG-I) and the translation efficiency is improved (ribosome binding capacity is enhanced). However, m1 ψ fails to completely eliminate immunogenicity, and it is still possible to activate innate immune responses through secondary pathways such as TLR3, leading to inflammatory side effects in some patients, and m1 ψ also has problems of translational fidelity, possibly causing ribosome misreading at high doses, leading to truncated proteins or abnormal translational slippage (Mulroney et al.,2024, nature).

Therefore, the development of a novel nucleotide modifier which can effectively avoid innate immune recognition, enhance translation activity and maintain translation fidelity is a key for breaking through the bottleneck of the current mRNA drug development.

Disclosure of Invention

The invention provides a deuterated nucleotide which can be used for in vitro transcription of mRNA, and can reduce inherent immunity and improve expression level and translation fidelity of mRNA encoding protein.

Nucleotide(s)

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

Wherein, the

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹ Each independently is H or D, provided that at least one of R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹ is D;

r ¹² is selected from

In some embodiments, at least one of R ⁵、R¹⁰、R¹¹ is D.

In some embodiments, R ⁵ is D.

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%.

In some embodiments, R ¹⁰、R¹¹ is D.

In some embodiments, R ¹⁰、R¹¹ 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 ¹⁰、R¹¹ has a deuterium content of at least 95%. In some embodiments, R ¹⁰、R¹¹ has a deuterium content of at least 98%.

In some embodiments, R ¹、R²、R³、R⁴、R⁶、R⁷、R⁸、R⁹ is H.

In some embodiments, R ¹² is

In some embodiments, the compound of formula (I) is selected from the group consisting of compounds of formula (I-1),

Wherein R ⁵、R¹⁰、R¹¹、R¹² is as defined in any embodiment of the invention.

In some embodiments, the nucleotide is selected from

Nucleoside

The second aspect of the present invention provides a nucleoside or a pharmaceutically acceptable salt thereof, wherein the nucleoside has a structure represented by formula (II)

Wherein ,R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹ is as defined in any one of the embodiments of the invention.

In some embodiments, the nucleoside is selected from

Polynucleotide

In a third aspect the invention 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 a nucleotide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof or a nucleoside according to the second aspect of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the modified uracil nucleotide or nucleoside comprises or has a structure of formula (III)

In some embodiments, the structure of formula (III) is selected from

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

In some embodiments, the structure of formula (III-1) is selected from

In some embodiments, each position of the nucleotide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof, the nucleotide according to the second aspect or a pharmaceutically acceptable salt thereof, the polynucleotide encoding the polypeptide and/or protein of interest according to the third aspect 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 as D has a deuterium content of at least 95%. In some embodiments, each position represented as D has a deuterium content of at least 98%.

In some embodiments, uracil nucleotides in the polynucleotides encoding the polypeptides and/or proteins of interest are replaced in whole or in part by a structure represented by formula (III) or formula (III-1).

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 guanylate, adenylate, cytidylate and a nucleotide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof. It will be appreciated by those skilled in the art that the nucleotides may be linked by linkages, for example, phosphodiester linkages.

Nucleic acid molecules

In a fourth aspect the invention provides a nucleic acid molecule comprising a polynucleotide encoding a polypeptide and/or protein of interest according to the third aspect of the invention.

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).

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

In some embodiments, the 5 'Cap structure is selected from 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.

In some embodiments, the nucleic acid molecule is selected from the group consisting of small interfering RNAs (sirnas), messenger RNAs (mrnas), short hairpin (shrnas), antisense oligonucleotides, and RNA aptamers.

In some embodiments, the nucleic acid molecule is mRNA.

In some embodiments, the nucleic acid molecule is isolated.

In some embodiments, the nucleic acid molecule is purified.

Carrier body

In a fifth aspect the invention provides a vector comprising a nucleic acid molecule according to the fourth aspect of the invention.

In some embodiments, the vector is selected from the group consisting of a plasmid, phage, and viral vector.

Delivery composition

In a sixth aspect the invention provides a delivery composition comprising a delivery vehicle and one or more selected from the group consisting of a polynucleotide encoding a polypeptide and/or protein of interest according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, and a vector according to the fifth aspect of the invention.

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 microbubble, a gene gun, and a viral vector (e.g., replication defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).

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

In some embodiments, the nanolipid particles comprise one or more of a cationic lipid, a pegylated lipid, a neutral lipid, and a steroid or steroid analog.

In some embodiments, the nanolipid particles comprise ionizable cationic lipid SM-102, helper lipid DSPC, cholesterol, and pegylated lipid PEG2000-DMG.

Pharmaceutical composition

The seventh aspect of the invention provides a pharmaceutical composition comprising a polynucleotide encoding a polypeptide and/or protein of interest according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention and/or a delivery composition according to the sixth aspect of the invention, and one or more pharmaceutically acceptable carriers and/or excipients.

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

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

The pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection and injectable concentrated solutions), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the pharmaceutical compositions of the present invention in the necessary amount in an appropriate solvent and optionally with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) and then filtering off. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as sterile pyrogen-free water.

Furthermore, the polynucleotides, nucleic acid molecules, vectors or delivery compositions of the invention encoding the polypeptides and/or proteins of interest may be present in the pharmaceutical composition in unit dosage form for ease of administration.

The pharmaceutical compositions of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. For many therapeutic uses, however, the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intratumoral). Those skilled in the art will appreciate that the route and/or mode of administration will vary depending upon the intended purpose. In a partially preferred embodiment, the pharmaceutical composition of the invention is administered by intravenous infusion or injection.

The pharmaceutical compositions of the invention may 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 may vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, such as age, weight and sex, the manner of administration of the drug, and other treatments administered simultaneously, and the like.

In the present invention, the dosing regimen may be adjusted to achieve the optimal target response (e.g., therapeutic or prophylactic response). For example, the dosage may be administered in a single dose, may be administered multiple times over a period of time, or may be proportionally reduced or increased as the degree of urgency of the treatment situation.

Kit for detecting a substance in a sample

In an eighth aspect, the invention provides a kit comprising a nucleotide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof.

In some embodiments, the kit further comprises Adenosine Triphosphate (ATP), guanosine Triphosphate (GTP), and Cytidine Triphosphate (CTP).

Use of the same

A ninth aspect of the invention provides the use of a nucleoside according to the second aspect of the invention or a pharmaceutically acceptable salt thereof in the preparation of a nucleotide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof.

The tenth aspect of the present invention provides the use of a nucleotide according to the first aspect of the invention or a pharmaceutically acceptable salt thereof, or a nucleoside according to the second aspect or a pharmaceutically acceptable salt thereof, or a kit according to the eighth aspect of the invention, for the preparation of a polynucleotide according to the third aspect of the invention encoding a polypeptide and/or protein of interest, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, a delivery composition according to the sixth aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention.

An eleventh aspect of the invention provides the use of a polynucleotide according to the third aspect of the invention encoding a polypeptide and/or protein of interest, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, a delivery composition according to the sixth aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition or for lessening the severity of said disease or condition.

A twelfth aspect of the invention provides a polynucleotide encoding a polypeptide and/or protein of interest according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, a delivery composition according to the sixth aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention for use in the treatment and/or prophylaxis of a disease or condition or for lessening the severity of said disease or condition.

A thirteenth aspect of the invention provides a method of treating and/or preventing a disease or disorder or reducing the severity of a disease or disorder, comprising administering to a subject an effective amount of a polynucleotide encoding a polypeptide and/or protein of interest according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention, a delivery composition according to the sixth aspect of the invention or a pharmaceutical composition according to the seventh aspect of the invention.

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 a supplemental dose of the polynucleotide, nucleic acid molecule, vector, delivery composition, or pharmaceutical composition.

In some embodiments, the subject is a mammal, e.g., a human.

Definition of terms

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "and/or" should be taken to mean a specific disclosure of each of two or more specified features or elements, as well as any combination of two or more features or elements. Thus, the term "and/or" as used in the phrase, for example, "a and/or B" is intended to include "a and B", "a or B", "a" (alone) and "B" (alone).

Unless otherwise indicated, when a position is designated as "H" or "hydrogen," or its chemical manifestation means hydrogen, it is understood to be hydrogen having a natural abundance isotopic composition.

Unless otherwise indicated, when a position is designated as "D" or "deuterium", it means 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%. Deuterium content can be measured by techniques known in the art, such as ¹ H NMR spectroscopy.

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

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the application that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include salts with inorganic or organic acids, or with acidic protons present on the parent compound replaced by metal ions, or with organic bases.

Pharmaceutically acceptable salts of the compounds of the invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups with inorganic acids (e.g., hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric) or with organic acids (e.g., acetic, oxalic, maleic, tartaric, citric, succinic or malonic) or by using other methods used in the art (e.g., ion exchange). Other pharmaceutically acceptable salts include adipic acid salts, alginates, ascorbates, aspartic acid salts, benzenesulfonic acid salts, benzoic acid salts, bisulfate salts, boric acid salts, butyric acid salts, camphoric acid salts, citric acid salts, cyclopentanepropionic acid salts, digluconate, dodecylsulfuric acid salts, ethanesulfonic acid salts, formic acid salts, fumaric acid salts, glucoheptonate, glycerophosphate, gluconic acid salts, hemisulfate, heptanoic acid salts, caproic acid salts, hydroiodides, 2-hydroxy-ethanesulfonic acid salts, lactobionic acid salts, lactic acid salts, lauric acid salts, lauryl sulfuric acid salts, malic acid salts, maleic acid salts, malonic acid salts, methanesulfonic acid salts, 2-naphthalenesulfonic acid salts, nicotinic acid salts, nitrate, oleic acid salts, oxalic acid salts, palmitic acid salts, pamoic acid salts, pectic acid salts, persulfates, 3-phenylpropionic acid salts, phosphate salts, pivalic acid salts, stearic acid salts, succinic acid salts, sulfuric acid salts, tartaric acid salts, thiocyanate salts, p-toluenesulfonic acid salts, undecanoic acid salts, valeric acid salts, and the like.

Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N ⁺(C_1-4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using, for example, halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate counter ions.

Pharmaceutically acceptable salts are also intended to cover semi-salts in which the ratio of compound to acid is 2:1, respectively. Exemplary hemi-salts are those derived from acids containing two carboxylic acid groups, such as malic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, glutaric acid, oxalic acid, adipic acid, and citric acid. Other exemplary semi-salts are those derived from a aprotic mineral acid (e.g., sulfuric acid). Preferred exemplary hemi-salts include, but are not limited to, hemi-maleate, hemi-fumarate, and hemi-succinate.

As used herein, the term "nucleic acid" is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester bonds. Nucleic acids may also encompass modified nucleic acid molecules, such as DNA or RNA molecules with base, sugar, or backbone modifications, for example deuterated modifications. The nucleic acid may be present in a variety of forms, such as an isolated segment of incorporated sequence and a recombinant vector or recombinant polynucleotide encoding a polypeptide, such as an antigen or one or both chains of an antibody, or a fragment, derivative, mutein or variant thereof, a polynucleotide sufficient for use as a hybridization probe, a PCR primer or a sequencing primer for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide.

As used herein, the term "polynucleotide" refers to a nucleic acid molecule that may be recombinant or that has been isolated from total genomic nucleic acid. In certain embodiments, the polynucleotide comprises regulatory sequences substantially separate from the naturally occurring gene or protein coding sequence thereof. The polynucleotide may be single-stranded (encoding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may (but need not) be present within the polynucleotide.

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, which are linked to each other along a so-called backbone. The backbone is formed by phosphodiester bonds 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, in cells by transcription of DNA sequences. In eukaryotic cells, transcription is usually performed in the nucleus or in the granulosa line. In vivo, transcription of DNA can produce immature RNA, which is processed into messenger RNA (mRNA). For example, processing of immature RNA in eukaryotic organisms involves various post-transcriptional modifications such as splicing, 5' capping, polyadenylation, export from the nucleus or centrosome. Processing mature messenger RNA and providing a nucleotide sequence that can be translated into an amino acid sequence of a peptide or protein. Mature mRNA can comprise a 5' cap, 5' UTR, open reading frame, 3' UTR, and polyadenylation tail sequence. The RNA may comprise all or most ribonucleotide residues. In one embodiment, the 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 will typically contain a5 'untranslated region (5' UTR), a polypeptide coding region, and a 3 'untranslated region (3' UTR). Without any limitation, RNA may encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, recombinantly produced RNA, and modified RNA.

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. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, xenogeneic homologs, fragments, and other equivalents, variants, and analogs of the foregoing. The polypeptide may be a single molecule or may be a multi-molecular complex, such as a dimer, trimer or tetramer. Proteins comprise one or more peptides or polypeptides and may be folded into a 3-dimensional form that may be required for the protein to perform its biological function.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, as 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 including, but not limited to: for example, pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic 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, osmotic pressure maintaining agents include, but are not limited to, sugar, naCl, and the like, absorption delaying agents include, but are not limited to, monostearates, and gelatin, diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), and the like, preservatives include, but are not limited to, various antibacterial and antifungal agents such as thiomersal, 2-phenoxyethanol, parabens, trichlorot-butanol, phenol, sorbic acid, and the like. Glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like.

As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease, and a disease-treating effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

As used herein, the term "treatment" is intended to alleviate, mitigate, ameliorate or eliminate a disease state or condition for which it is intended. A subject is successfully "treated" if one or more of the subject's indications and symptoms exhibit an observable and/or detectable decrease or improvement. It is also to be understood that the treatment of the disease state or condition includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant result.

As used herein, the term "preventing" is intended to avoid, reduce, prevent or delay the appearance of a disease or disease-related symptoms, and the absence of such disease or disease-related symptoms prior to administration of the relevant drug. "preventing" is not required to completely prevent the occurrence of a disease or disease-related symptom, e.g., a subject may be reduced in risk of developing a particular disease or disease-related symptom after administration of a related agent, or may be considered "preventing" the occurrence or progression of the disease by reducing the severity of the related symptom that later develops.

Advantageous effects

The deuterated nucleotide provided by the invention can improve the level of protein expressed by mRNA, reduce inherent immune activation and increase translation fidelity, so that the optimized mRNA molecule has wide application prospects in the fields of vaccines, gene therapy, antibody therapy and the like.

Drawings

FIG. 1. Electrophoresis of chemically modified mRNA (1).

FIG. 2 toxicity test results of chemically modified mRNA in cells.

FIG. 3. Results of parameter detection of chemically modified mRNA lipid nanoparticles.

FIG. 4 shows the results of expression of chemically modified mRNA in mice.

FIG. 5 intrinsic immune response levels of chemically modified mRNA in mice.

FIG. 6 shows electrophoresis of chemically modified mRNA (2).

FIG. 7 shows the result of translation shift rate detection of chemically modified mRNA.

Wherein, in the figure, p <0.05, p <0.01 and p <0.001 are shown.

Sequence information

The information of the partial sequences according to the present application is shown in table 1 below.

TABLE 1 information on partial sequences

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example one preparation of ((2R, 3S,4R, 5R) -3, 4-dihydroxy-5- (5-methoxy-2, 4, -dioxo-3, 4-dihydropyrimidin-1 (2H) -yl-6-d) tetrahydrofuran-2-yl) methyltetrahydrophosphate (2583U)

Step 1) 5-methoxypyrimidine-2, 4 (1H, 3H) -dione-6-d

Sodium metal (138 mg,6.0 mmol) was added in portions to heavy water (2.0 mL) under ice and stirred for 30 min. Then 5-methoxyuracil (265 mg,1.87 mmol) was added and the system was replaced with argon and the tube was sealed and heated to 125℃for 16 hours. After the reaction solution cooled to room temperature, acidizing with 1.0N hydrochloric acid, filtering, collecting a filter cake, and drying to obtain the title product, 240mg of white solid, and the yield is 90%. LC-MS (ESI) m/z=166.1 [ M+Na ⁺ ].

Step 2) (2R, 3S,4R, 5R) -2- ((benzoyloxy) methyl) -5- (5-methoxy-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl-6-d) tetrahydrofuran-3, 4-dibenzoate

To a solution of 5-methoxypyrimidine-2, 4 (1H, 3H) -dione-6-d (240 mg,1.68 mmol) in anhydrous acetonitrile (20 mL) was added N, O-bis-trimethylsilylacetamide (BSA) (1.25 mL,5.04 mmol), and the resulting mixture was heated at reflux under argon for 1 hour. Cooled to room temperature, 1-acetyl-tris-benzyloxy-primary saccharide (1.1 g,2.18 mmol)) and trimethylsilyl triflate (0.40 mL,2.18 mol) were added. The reaction mixture was heated to reflux for 2 hours. After the reaction solution was cooled to room temperature, dried under reduced pressure, and the obtained 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 ⁺ ].

Step 3) 1- ((2R, 3R,4S, 5R) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5-methoxypyrimidin-2, 4 (1H, 3H) -dione-6-d

(2R, 3S,4R, 5R) -2- ((benzoyloxy) methyl) -5- (5-methoxy-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl-6-d) tetrahydrofuran-3, 4-dibenzoate (370 mg,0.63 mmol) was added to 7N methanolic ammonia solution (6 mL)) and reacted at 45℃for 24 hours. Spin-drying under reduced pressure, and pulping the residue with ethyl acetate and acetonitrile sequentially to obtain the title product, 145mg of white solid, and yield ：83％.LC-MS(ESI):m/z＝276.0[M+H⁺].¹H-NMR(500MHz,CD₃OD)δ5.98-5.95(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).

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) methyltetrahydrophosphate (2583U)

To a solution of 1- ((2R, 3R,4S, 5R) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione-6-d (50 mg,0.182 mmol) in trimethyl phosphate (2 mL) under argon at 0℃was added dropwise phosphorus oxychloride (25. Mu.L, 0.273 mmol) and the resulting mixture stirred for half an hour. Phosphorus oxychloride (25 μl,0.273 mmol) was added and stirred for 1 hour. A solvent of 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. Diluting with water to 30mL, purifying with 650Q ion column (eluent: 1M triethylammonium bicarbonate buffer), collecting product tube, concentrating, purifying with reverse column for three times, and purifying the obtained product with 650Q ion column (eluent: 1M triethylammonium bicarbonate buffer) to obtain triethylamine salt 60mg of the title product with yield of 32%. Deuteration rate is as follows 98％.¹H NMR(500MHz,D₂O)δ6.07(s,1H),4.47(s,2H),4.32-4.28(m,2H),4.24-4.22(m,1H),3.83(s,3H).³¹P NMR(202MHz,D₂O)δ-10.80(d,J＝19.6Hz,1P),-11.74(d,J＝19.9Hz,1P),-22.30(t,J＝19.3Hz,1P).

EXAMPLE two preparation of ((2R, 3S,4R, 5R) -3, 4-dihydroxy-5- (5-methoxy-2, 4, -dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) tetrahydrofuran-2-yl) methyl-d ₂ tetrahydrotriphosphate (2586U)

Step 1) 1- ((3 aR,4R,6 aR) -6- (hydroxymethyl) -2, 2-dimethyltetrahydrofurano [3,4-d ] [1,3] dioxan-4-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione

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, stirred for 10 min, then dried by spin-drying and purified by column chromatography (dichloromethane: methanol=50:1 to 25:1) to give the title product as a solid 2.52g in 92% yield. L-MS (ESI) m/z=314.9 [ M+H ] ⁺.

Step 2) (3 aS,4S,6R,6 aR) -6- (5-methoxy-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2, 2-dimethyltetrahydrofurano [3,4-d ] [1,3] dioxane-4-carboxylic acid tert-butyl ester

To a solution of 1- ((3 aR,4R,6 aR) -6- (hydroxymethyl) -2, 2-dimethyltetrahydrofurano [3,4-d ] [1,3] dioxan-4-yl) -5-methoxypyrimidine-2, 4 (1H, 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. Spin-drying and column chromatography purification (petroleum ether: ethyl acetate=2:1 to 1:1) gave the title product as a solid 1.8g in 63% yield. LC-MS (ESI) m/z=384.9 [ M+H ] ⁺.

Step 3) 1- ((3 aR,4R,6 aR) -6- (hydroxymethyl-d ₂) -2, 2-dimethyltetrahydrofurano [3,4-d ] [1,3] dioxan-4-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione

Sodium borodeuteride (304 mg,7.26 mmol) was added portionwise to a solution of tert-butyl (3 as,4s,6r,6 ar) -6- (5-methoxy-2, 4-dioxo-3, 4-dihydropyrimidin-1 (2H) -yl) -2, 2-dimethyltetrahydrofurano [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) under ice-bath. After the addition was completed, the reaction was allowed to proceed to 50℃for 4 hours. The pH was adjusted to about 7 with deuterated acetic acid, water was added, extraction was performed with ethyl acetate, the organic layers were combined, washed with saturated saline, dried over anhydrous sodium sulfate, and spin-dried, and the resulting residue was purified by column chromatography (petroleum ether: ethyl acetate=1:1 to 0:1) to give the title product as a white solid, 420mg, yield: 55%. LC-MS (ESI) m/z=317.0 [ M+H ] ⁺.

Step 4) 1- ((2R, 3R,4S, 5R) -3, 4-dihydroxy-5- (hydroxymethyl-d ₂) tetrahydrofuranyl-2-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione

1- ((3 AR,4R,6 aR) -6- (hydroxymethyl-d ₂) -2, 2-dimethyltetrahydrofurano [3,4-d ] [1,3] dioxan-4-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione (420 mg,1.33 mmol) was dissolved in trifluoroacetic acid (3 mL) and water (3 mL) and reacted at room temperature for 2 hours. Spin-drying, dissolving with ethanol, spin-drying, and pulping with ethyl acetate to obtain the title product as white solid 349mg with a yield of 95%. LC-MS (ESI) m/z=276.9 [ M+H ] ⁺.

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-d ₂ tetrahydrotriphosphate (2586U)

Referring to example one, 1- ((2R, 3R,4S, 5R) -3, 4-dihydroxy-5- (hydroxymethyl-d ₂) tetrahydrofuran-2-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione is used instead of 1- ((2R, 3R,4S, 5R) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5-methoxypyrimidine-2, 4 (1H, 3H) -dione-6-d to give 40mg of the triethylamine salt of the title product in 27% yield. Deuteration rate is as follows 98.5％.MS(ESI):m/z＝515.0[M-H]^-.¹H NMR(500MHz,D₂O)δ7.41(s,1H),6.08(d,J＝5.5Hz,1H),4.51-4.48(m,2H),4.34(s,1H),3.85(s,3H).³¹P NMR(202MHz,D₂O)δ-10.21(d,J＝19.1Hz,1P),-11.67(d,J＝20.0Hz,1P),-23.18(t,J＝19.9Hz,1P).

Example III In Vitro Transcription (IVT)

A linearized template for IVT was prepared using PCR method, performed with Renzan 2X PHANTA FLASH MASTER Mix. The PCR reaction was carried out in the following system under the reaction conditions of 98℃for 5 seconds, 25 cycles of 98℃for 10 seconds, 58℃for 5 seconds, 72℃for 10 seconds, 72℃for 1 minute, and 4℃for termination.

TABLE 2PCR reaction system (50. Mu.L)

PCR amplification was performed using EGFP (SEQ ID NO: 1) and Fluc (Firefly Luciferase ) (SEQ ID NO: 2) as plasmid templates, respectively. The amplification primer sequence of the plasmid template is as follows:

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

HBB-R:

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAATGAAAATAAATGTTTTTTATTAGG(SEQ ID NO:6).

IVT templates (SEQ ID NO:1 and SEQ ID NO: 2) in the PCR products were recovered using the Promega (cat# A9282) kit.

Using natural nucleotides as well as modified nucleotides to transcribe into mRNA, the IVT system was configured as follows:

TABLE 3IVT reaction system (20. Mu.L)

Wherein, the NTPs of the control group are composed of natural nucleotide ATP, GTP, CTP, UTP, the NTPs of the experiment group 1 are composed of natural nucleotide ATP, GTP, CTP and modified nucleotide 2583U, and the NTPs of the experiment group 2 are composed of natural nucleotide ATP, GTP, CTP and modified nucleotide 2586U. IVT reactions were performed using EGFP (SEQ ID NO: 1) and Fluc (SEQ ID NO: 2) as templates, respectively, as follows.

After addition of the template, incubation was carried out for 2h at 37℃and 1. Mu.L DNaseI was added to each reaction and incubated at 37℃for 30min to digest the DNA template, and RNA was precipitated at-80℃by LiCl precipitation. Centrifuging to collect settled mRNA, centrifuging at 14,000rpm for 10min at 4 ℃ to obtain white precipitate of RNA at the bottom of a centrifuge tube, removing supernatant, adding 1mL of precooled 70% ethanol, washing RNA by vortex, repeating washing for 1 time, drying at room temperature until the precipitate becomes semitransparent, adding RNase-free water to fully dissolve RNA, measuring mRNA concentration by Nanodrop One, and detecting mRNA quality by 1% TBE agarose gel electrophoresis. As a result, as shown in FIG. 1, mRNA products of EGFP (SEQ ID NO: 1) and Fluc (SEQ ID NO: 2) of higher quality were produced by substituting the modified nucleotides 2583U, 2586U for the natural UTP, respectively.

Example IV toxicity detection of chemically modified mRNA in cells

Toxicity of chemically modified mRNA was detected in a549 cells. A96-well plate was inoculated with 100. Mu.L per well at a concentration of 0.3X10 ⁶/mL using DMEM medium containing 10% fetal bovine serum and 1% penicillin. After 12 hours of incubation, lipo8000 and mRNA prepared in example three were transfected into the A549 cell line at a concentration of 1.6:1 at 1ug/mL using opti-MEM. And a control cell without any treatment, a lipo8000 group added only at 0.16. Mu.L/well, and a poly (I: C) group transfected with lipo8000 and poly (I: C) at a concentration of 1.6:1 at 1ug/mL were set. The transfected cells were placed in a 37℃cell incubator for 48h and tested using a microplate reader for Cell Counting Kit-8. The results are shown in FIG. 2, where the chemically modified mRNA was non-toxic to A549 cells.

EXAMPLE five expression of chemically modified mRNA in mice

SM102, DSPC, cholesterol and DMG-PEG2000 were mixed in ethanol at a molar ratio of 50:10:38.5:1.5, and then the lipid mixture was mixed with the Fluc mRNA prepared in example three (dissolved in 50mM sodium acetate pH 4.0) at a volume ratio of 1:3 using a microfluidic mixer, setting a total flow rate of the two mixtures of 12mL/min, and a flow rate ratio of 1:3. And (3) removing acetic acid and ethanol in the mRNA-LNP by using PBS for 4 hours, changing the PBS by using PBS for 2 hours, and obtaining the Fluc-mRNA-LNP by using the PBS to be dialyzed to be neutral. Particle size and distribution of the Fluc-mRNA-LNP were measured by a nanoparticle size analyzer (Malvern), and encapsulation efficiency of mRNA was measured by Ribogreen method (Thermo Fisher), and as shown in fig. 3, the average particle size of the encapsulated vaccine was 60 to 80nm, the particle size distribution was uniform (PDI < 0.1), the encapsulation efficiency was more than 90%, and the quality was good.

BALB/c female 6 week old mice were randomly grouped in 5/group, fluc-mRNA-LNP was intramuscular injected at a dose of 5 μg/group, and after 6 hours, luciferase (PERKIN ELMER) substrate was injected at 100 uL/group for in vivo imaging using a AniView100 Pro multimode animal in vivo imaging system. As a result, as shown in FIG. 4, in the BALB/c mouse, the Fluc mRNA expression amount of the modified nucleotide 2586U was not significantly changed, while the Fluc mRNA expression amount of the modified nucleotide 2583U was significantly reduced, as compared with UTP.

EXAMPLE six innate immune response of chemically modified mRNA in mice

BALB/C female 6-week-old mice were randomly grouped into 5 groups at a dose of 5 μg/min, and the Fluc-mRNA-LNP prepared in example five was intramuscular injected with PBS at a dose of 100 μl/min, and a negative control group was set, and the positive control group was intramuscular injected with poly (I: C) at a dose of 5 μg/min, spleen was collected after 6 hours, and mRNA levels of IFNb were quantified using RNAiso Plus extraction RNA, hyperScript III RT SuperMix for QPCR WITH GDNA reverse transcription, 2x S6 Universal SYBR qPCR Mix and QuantStudio ^TM Flex. As a result, as shown in FIG. 5, in BALB/c mice, IFNb mRNA levels produced by the stimulation of Fluc mRNA of modified nucleotide 2586U were significantly reduced compared to UTP, and IFNb mRNA levels produced by the stimulation of Fluc mRNA of modified nucleotide 2583U were not significantly different.

Example seven chemically modified mRNA translation Shift rate detection

To examine the effect of the modified nucleotide on translational fidelity, a CTP was inserted after nucleotide 681-684 (TTTT) of Fluc-WT-2 (SEQ ID NO: 3) to obtain reporter gene Fluc-ISF-2 (SEQ ID NO: 4) for detecting translational frameshift. Only when the ribosome moves frame (FRAME SHIFT) at 4 consecutive UTPs across subsequent CTPs, can Fluc-ISF-2 express a functional Fluc, the expression ratio of Fluc-ISF-2 to Fluc-WT-2 representing the frequency of frame movement. mRNA of Fluc-WT-2 and Fluc-ISF-2 was prepared as in example three using the amplification primer sequences (SEQ ID NO: 11) and (SEQ ID NO: 12), and the results are shown in FIG. 6.

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

HBB-R'：TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCGAGAGCGTAATCTGGAAC(SEQ ID NO:12).

Chemically modified Fluc-WT-2 and Fluc-ISF-2mRNA were transfected with lipo8000 into HeLa cells and after 24 hours of incubation fluke luciferase reporter gene assay kit (next holt) and PERKIN ELMER multifunctional microplate reader were used to quantify Fluc-WT-2 and Fluc-ISF-2mRNA expression. As shown in fig. 7, in HeLa cells, mRNA prepared from modified nucleotide 2586U was significantly reduced in rate of shift compared to mRNA prepared from modified nucleotide 2583U.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the specific embodiments of the present invention may be modified or some technical features may be equivalently replaced, and they are all included in the scope of the technical solution of the present invention as claimed.

Claims

1. A nucleotide or a pharmaceutically acceptable salt thereof, wherein the nucleotide has a structure represented by formula (I)

Wherein, the

r ¹² is selected from

2. The nucleotide or pharmaceutically acceptable salt thereof according to claim 1, wherein at least one of R ⁵、R¹⁰、R¹¹ is D;

preferably, R ⁵ is D;

preferably, R ¹⁰、R¹¹ is D.

3. The nucleotide or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the nucleotide is selected from the group consisting of

4. The nucleotide or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein each position represented as D has a deuterium content of at least 70%;

Preferably, each position represented as D has a deuterium content of at least 95%;

preferably, each position represented as 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 ,R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹ is as defined in any one of claims 1 to 4.

6. The nucleoside or pharmaceutically acceptable salt thereof of claim 5, wherein the nucleoside is selected from the group consisting of

7. The nucleoside or pharmaceutically acceptable salt thereof of claim 5 or 6, wherein each position represented as D has a deuterium content of at least 70%;

8. Use of a nucleoside according to any one of claims 5 to 7, or a pharmaceutically acceptable salt thereof, for the preparation of a nucleotide according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof.

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 a nucleotide or pharmaceutically acceptable salt thereof according to any of claims 1-4 or a nucleoside or pharmaceutically acceptable salt thereof according to any 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 the structure of formula (III)

Wherein ,R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹ is as defined in any one of claims 1 to 4;

Preferably, the structure of formula (III) is selected from

Preferably, each position represented as D has a deuterium content of at least 70%;

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' poly-a tail;

preferably, the nucleic acid molecule comprises, in order from the 5' end to the 3' end, a 5' cap structure, a 5' UTR, a 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 oligonucleotides and RNA aptamers;

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 the group consisting of plasmid, phage and viral vectors.

14. A delivery composition comprising a delivery vehicle selected from one or more of the polynucleotides encoding a polypeptide and/or protein of interest according to claim 9 or 10, the nucleic acid molecules according to claim 11 or 12, the vectors according to claim 13;

Preferably, the delivery vehicle is selected from the group consisting of lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microbubbles, gene-guns, and viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses);

preferably, the delivery vehicle is a nanolipid particle;

preferably, the nanolipid particles comprise one or more of cationic lipids, pegylated lipids, neutral lipids, and steroids or steroid analogues;

preferably, the nano-lipid particles comprise the ionizable cationic lipid SM-102, the helper lipid DSPC, cholesterol and the pegylated lipid PEG2000-DMG.

15. A pharmaceutical composition comprising a polynucleotide encoding a polypeptide and/or protein of interest according to claim 9 or 10, a nucleic acid molecule according to claim 11 or 12, a vector according to claim 13 and/or a delivery composition according to 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, nucleic acid molecule, vector and/or delivery composition encoding the polypeptide and/or protein of interest.

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

18. Use of a polynucleotide encoding a polypeptide and/or protein of interest according to claim 9 or 10, a nucleic acid molecule according to claim 11 or 12, a vector according to claim 13, a delivery composition according to claim 14 or a pharmaceutical composition according to claim 15 for the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition or for lessening the severity of said disease or condition.