Oligonucleotides having modified nucleotide residues at 5 '-terminus and uses thereof

Abstract

The invention relates to the field of biological medicine, in particular to oligonucleotide with modified nucleotide residues at the 5'terminal and application of the oligonucleotide. The invention provides a uracil derivative which is uracil modified by an aromatic compound, and the aromatic compound is selected from thiophene, pyridine or phenylpropionic acid. The invention provides an oligonucleotide, the 5'terminal of the oligonucleotide is a residue with a modified basic group, and the modification is selected from thiophene modification, pyridine modification or phenylpropionic acid modification. The invention also provides an oligonucleotide conjugate and a pharmaceutical composition related to the oligonucleotide, and application of the oligonucleotide conjugate and the pharmaceutical composition in preparation of nucleic acid drugs. Specific structural modification is carried out on the 5'tail end of oligonucleotide, so that the affinity of oligonucleotide and AGO2 is improved, the silencing efficiency of a drug on a target gene is improved, and diseases and symptoms related to the expression level of the target gene can be prevented and treated.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and in particular to oligonucleotides having modified nucleotide residues at the 5' end and uses thereof. Background Art

[0002] Small interfering RNA (siRNA) is an emerging therapeutic modality. siRNA forms an RNA-induced silencing complex (RISC) with proteins such as Argonaute-2 (AGO2). The siRNA separates into two single strands: a guide strand (the antisense strand) and a passenger strand (the sense strand). The guide strand specifically recognizes and cleaves disease-associated mRNA, thereby directly blocking the production of disease-associated proteins at the translational level and exerting a therapeutic effect. The AGO2 protein is a key component in cleaving target mRNAs. It has three functional domains: the PAZ domain, the MID domain, and the PIWI domain. The MID domain can bind to the 5′ end of nucleic acids.

[0003] siRNAs are small RNAs (siRNAs) approximately 21 nt in length. Under the action of the nuclease Argonaute 2, they bind to the entire strand of the target gene's mRNA, efficiently cleaving it and inhibiting gene expression. To date, six siRNAs are commercially available, all targeting liver cells. They offer long-lasting efficacy with a semi-annual injection schedule, and their therapeutic efficacy significantly surpasses that of small molecule drugs. Two key factors underpin the superior performance of these marketed siRNAs: first, modification with 2'-O-methyl, 2'-deoxy-2'-fluoro, and phosphorothioates significantly enhances the siRNA's resistance to degradation by various RNases, improving its stability while preserving the gene-silencing activity of the siRNA antisense strand. Second, a cleavable linker is used to conjugate N-acetylgalactosamine to the 3' end of the siRNA sense strand, specifically binding to the ASGPR receptor, which is abundant on the liver cell membrane. This results in highly specific liver cell targeting, with over 60% of siRNA molecules entering the liver, ensuring a sufficient number of siRNA molecules in the liver. Therefore, the superior liver pharmacokinetic characteristics, good intracellular stability and gene silencing ability of GalNAc-coupled full-chain modified siRNA are the key factors for the liver-targeted siRNA to achieve efficient gene silencing for more than 6 months.

[0004] Currently, siRNA is used in the treatment of both liver and extrahepatic diseases. Its mechanism of action inside and outside the liver is the same, both of which rely on the affinity of the 5' nucleotide. The application of siRNA in the liver includes liver diseases, such as non-alcoholic fatty liver disease (NAFLD), cirrhosis, and liver cancer. The application of siRNA outside the liver includes drugs for the treatment of central nervous system diseases, eye diseases, kidney diseases, respiratory diseases, cancer, vascular diseases, metabolic diseases, autoimmune diseases, inflammatory bowel disease, genetic diseases, infectious diseases, respiratory diseases, liver diseases, kidney diseases, eye diseases, bone diseases, mental illnesses, blood diseases, skin diseases, etc., such as Parkinson's disease, Alzheimer's disease, Huntington's disease, spinal lateral sclerosis, myasthenia gravis, dry eye, optic atrophy, glaucoma, age-related macular degeneration, etc. However, the application of siRNA is still limited by the lack of an efficient delivery system similar to GalNAc. It requires the use of fatty chain modification, LNP encapsulation, etc., combined with local administration routes such as intrathecal injection to achieve long-term gene silencing of about 3 months, but the dosage is higher and the side effects are more serious. In addition, in some siRNAs used outside the liver, the affinity of the 5-terminal nucleotide to AGO2 is enhanced by modifying the monophosphate of the 5'-E-VP of the siRNA antisense chain, thereby improving the gene silencing ability and stability of the siRNA itself. However, in diseases such as tumors and autoimmune diseases that cannot be treated by targeting the liver or local injection, the aforementioned combined fatty chain and 5'-E-VP modified siRNA cannot effectively silence the target gene in vivo, and the long-term effect cannot be reflected. Therefore, new solutions to enhance the affinity of the 5'-terminal nucleotide to AGO2 need to be developed. Summary of the Invention

[0005] In order to address the deficiencies of the prior art, the present application modifies the 5' end of the siRNA antisense chain to enhance its affinity with AGO2 and better improve its gene silencing ability, which will effectively promote the application of siRNA in various diseases related to the mRNA level of gene expression inside and outside the liver.

[0006] In a first aspect, the present application provides a uracil derivative, which is uracil modified with an aromatic compound, wherein the aromatic compound is selected from thiophene, pyridine, or phenylpropionic acid.

[0007] The uracil derivative is a compound having a structure represented by any one of the following formulas I to VI, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a racemate thereof:

[0008]

[0009]

[0010]

[0011] Wherein, R1 is independently selected from OH or OMe, R2 is independently selected from OH or The second aspect of the present application provides the use of the uracil derivative of the first aspect, wherein the use is selected from:

[0012] 1) as the 5' end of the oligonucleotide;

[0013] 2) Improve the gene silencing ability of oligonucleotides or the RNA-induced silencing complexes they form.

[0014] Preferably, the oligonucleotide is siRNA; more preferably, the oligonucleotide is the antisense strand of siRNA.

[0015] The third aspect of the present application provides a method for modifying an oligonucleotide, which uses the uracil derivative according to the first aspect as the 5' end of the oligonucleotide.

[0016] The fourth aspect of the present application provides a method for improving the gene silencing ability of an oligonucleotide or an RNA-induced silencing complex formed therefrom, which comprises using the uracil derivative of the first aspect as the 5' end of the oligonucleotide.

[0017] In a fifth aspect, the present application provides an oligonucleotide, wherein the 5' end of the oligonucleotide is the uracil derivative according to claim 1 or 2.

[0018] Preferably, the 5' end of the oligonucleotide has a structure represented by any one of the following formulas VII to XII:

[0019]

[0020]

[0021]

[0022] wherein R1 is selected from OH or OMe.

[0023] Preferably, R1 is OMe.

[0024] In a sixth aspect, the present application provides an oligonucleotide conjugate, which contains the oligonucleotide according to the fifth aspect and a conjugation group connected to the oligonucleotide.

[0025] In a seventh aspect, the present application provides a pharmaceutical composition comprising the oligonucleotide according to the fifth aspect, or the oligonucleotide conjugate according to the sixth aspect.

[0026] The eighth aspect of the present application is the use of the uracil derivative of the first aspect, or the oligonucleotide of the fifth aspect, or the oligonucleotide conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect in the preparation of nucleic acid drugs or target protein expression inhibitors.

[0027] Preferably, the nucleic acid drug is a siRNA drug.

[0028] More preferably, the nucleic acid drug is a drug for a disease related to the mRNA level expressed by the target gene.

[0029] More preferably, the nucleic acid drug is a drug for preventing and / or treating any one or more of the following diseases: liver disease, central nervous system disease, eye disease, kidney disease, respiratory disease, cancer, vascular disease, metabolic disease, autoimmune disease, inflammatory bowel disease, genetic disease, infectious disease, respiratory disease, liver disease, kidney disease, eye disease, bone disease, mental illness, blood disease, skin disease.

[0030] The ninth aspect of the present application provides a method for regulating the expression level of a target gene in a cell in vitro, comprising administering to the cell an effective amount of the oligonucleotide of the fifth aspect, or the oligonucleotide conjugate of the sixth aspect, or the pharmaceutical composition of the seventh aspect.

[0031] The beneficial effects of this application are:

[0032] The present application performs specific structural modification on the 5' end of the oligonucleotide, thereby increasing the affinity of siRNA to AGO2 and improving the silencing efficiency of the siRNA drug on the target gene, which can prevent and treat diseases and conditions related to the expression level of the target gene, improve the effect and durability of the treatment, and the modified siRNA of the present application is biosafe and has an extremely low risk of genomic insertion. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The affinity of UMP, 001, 002 and 003 monophosphate to hAGO2-MID was quantified by 2D-HSQC NMR in Example 4; wherein, Figure 1 A is the migration spectrum, Figure 1 B is the affinity detection test result.

[0034] Figure 2 The crystal structure of the hAGO2-MID domain in complex with 001, 002 and 003 monophosphates in Example 5; wherein, Figure 2A is the overall structure of the non-natural nucleoside monophosphate in the hAGO2-MID binding pocket. The theme colors of the complexes of UMP, 001, 002 and 003 monophosphate with hAGO2-MID are gray, light pink, light orange and light blue, respectively. The skeleton of hAGO2-MID is shown as a cartoon model, the key amino acid residues that stabilize NMP are shown as stick models, water molecules are represented by sea blue spheres, potential hydrogen bonds are represented by black dotted lines, and the level of the electron density contours of 001, 002, and 003 monophosphates is adjusted to 2.0σ and represented by light green grids. Figure 2 (B) Overlay of UMP, 001, 002, and 003 monophosphates in the hAGO2-MID complex. Only the bulk of the UMP complex with hAGO2-MID is shown as a gray cartoon model, with the other three models aligned to it. Water molecules surrounding the cavity are colored according to the color of each motif defined in (A).

[0035] Figure 3 This is the in vitro and in vivo activity evaluation of the siRNA modified with 001m to 003m in Example 6; wherein, Figure 3 A is the RISC loading efficiency of siRNA bound to AGO2 protein, Figure 3 B is the IC50 value of siRNA in HepG2 cells, Figure 3 C is the residual ANGPTL3 mRNA in monkey primary hepatocytes after siRNA administration, Figure 3 D shows the residual human ANGPTL3 mRNA in the liver of mice after siRNA administration.

[0036] Figure 4 is the genome insertion risk of siRNA modified with 001m to 003m in Example 8; wherein, Figure 4 A is the content of aristolochic acid I-DNA adducts in mouse liver DNA, Figure 4 B is the potential incorporation of 003m into mouse liver DNA after administration of 003m-modified siRNA. Figure 4 C is the gene silencing effect of human ANGPTL3 mRNA in the liver of AAV8-TBG-ANGPTL3 infected mice after administration of 003m modified siRNA, Figure 4 D is the potential incorporation of 003m into total RNA of mouse liver after administration of 003m-modified siRNA. DETAILED DESCRIPTION

[0037] The following describes the embodiments of the present application through specific examples. Those skilled in the art can easily understand the other advantages and effects of the present application from the contents disclosed in this specification. The present application can also be implemented or applied through different specific embodiments, and the details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present application.

[0038] Before further describing the specific embodiments of the present application, it should be understood that the scope of protection of the present application is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present application are for describing specific specific embodiments rather than for limiting the scope of protection of the present application; in the specification and claims of the present application, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0039] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified herein, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of this application, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present application can also be used to realize this application.

[0040] The present application modifies the 5' end of the oligonucleotide, more specifically, modifies the base of the nucleotide at the 5' end of the oligonucleotide, so that the 5' end of the oligonucleotide is a non-natural nucleotide, thereby improving its silencing effect on the target gene.

[0041] The present application first provides a uracil derivative, which is uracil modified with an aromatic compound, wherein the aromatic compound is selected from thiophene, pyridine, or phenylpropionic acid.

[0042] The thiophene modification refers to the process of introducing a thiophene group (thiophene) or its derivatives into a chemical molecule. Thiophene is a five-membered heterocyclic compound containing a sulfur atom.

[0043] The pyridine modification refers to the process of introducing a pyridine ring or its derivatives into a chemical molecule. Pyridine is a six-membered heterocyclic compound containing one nitrogen atom.

[0044] The phenylpropanoic acid modification is the process of introducing phenylpropanoic acid or its derivatives into a chemical molecule. Phenylpropanoic acid is an aromatic carboxylic acid composed of a benzene ring and a propionic acid group (-CH2-CH2-COOH).

[0045] The uracil derivative is a compound having a structure represented by any one of the following formulas I to VI, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a racemate thereof:

[0046]

[0047]

[0048] Wherein, R1 is independently selected from OH or OMe (methoxy); R2 is independently selected from OH or

[0049] The pharmaceutically acceptable salts refer to salts of the compounds provided herein, which are prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of the compound of the present invention with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases, and the salts of the inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, and the like. The salts of the organic non-toxic bases include salts of primary, secondary, and tertiary amines, including substituted amines and cyclic amines. For example: N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, guaiac, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc. When the compound contains a relatively basic functional group, the acid addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of acid in a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid, and the like.

[0050] Stereoisomers refer to compounds whose atoms have the same order of arrangement in a molecule but different spatial arrangements, resulting in different structures in three-dimensional space and thus different physical and chemical properties. Stereoisomers can be divided into enantiomers, cis-trans isomers, configurational isomers, geometric isomers, and optical isomers; optical isomers include R-isomers and S-isomers.

[0051] The racemate refers to an equimolar mixture of a chiral molecule with optical activity (see optical isomerism) and its enantiomer.

[0052] The present application also provides uses of the above-mentioned uracil derivatives, which are selected from:

[0053] 1) as the 5' end of the oligonucleotide;

[0054] 2) Improve the gene silencing ability of oligonucleotides or the RNA-induced silencing complexes they form.

[0055] In a specific embodiment of the present application, the oligonucleotide may be siRNA, and the siRNA may include a sense strand and an antisense strand; preferably, the oligonucleotide may be the antisense strand of the siRNA.

[0056] In a specific embodiment of the present application, the above-mentioned uracil derivative can be used as the 5' end of the oligonucleotide chain, for example, as the raw material to synthesize the 5' end of the oligonucleotide chain, so that the 5' end of the oligonucleotide chain is a uracil nucleotide residue with a thiophene modification, or a pyridine modification, or a phenylpropionic acid modification.

[0057] In a specific embodiment of the present application, the above-mentioned uracil derivatives can be used to enhance the gene silencing ability of oligonucleotides or RNA-induced silencing complexes formed therefrom.

[0058] The RNA-induced silencing complex (RISC) formed by the complex refers to a multiprotein complex that plays a key role in the RNA interference (RNAi) process. It is composed of small RNAs (such as siRNA or miRNA), Argonaute proteins, and other auxiliary factors (such as Dicer enzyme, TRBP, etc.). siRNA binds to Argonaute proteins to form a pre-RISC (pre-RISC). Under the action of ATP-dependent helicases, the siRNA double strands unwind, with one strand (the guide strand) being retained and the other strand being discarded. The activated RISC localizes to the target mRNA homologous to the siRNA sequence through complementary base pairing with the guide strand.

[0059] The gene silencing ability refers to the ability to inhibit or shut down the expression of the target gene through a specific technology or mechanism.

[0060] In a specific embodiment of the present application, when the above-mentioned uracil derivative is used as the 5' end of an oligonucleotide chain, for example, as a raw material to synthesize the 5' end of an oligonucleotide chain, so that the 5' end of the oligonucleotide chain is a uracil nucleotide residue with a thiophene modification, or a pyridine modification, or a phenylpropionic acid modification as the base, the ability and efficiency of the oligonucleotide to recognize, bind to, and silence the target gene can be improved.

[0061] The present application also provides a method for modifying an oligonucleotide, which uses the above-mentioned uracil derivative as the 5' end of the oligonucleotide, and the oligonucleotide can be the antisense strand of siRNA.

[0062] In a specific embodiment of the present application, the modification method can be to first synthesize the above-mentioned uracil derivatives and other nucleotides, and then use the uracil derivatives as raw materials to connect to the 5' end of the oligonucleotide chain through reaction, and the 3-position oxygen atom of the uracil derivatives as raw materials after the reaction is connected to the adjacent nucleotide residue, for example, a solid phase synthesis method can be used.

[0063] The present application also provides a method for improving the gene silencing ability of an oligonucleotide or an RNA-induced silencing complex formed therefrom, which comprises using the above-mentioned uracil derivative as the 5' end of the oligonucleotide, and the oligonucleotide may be the antisense strand of siRNA.

[0064] In a specific embodiment of the present application, the method for improving the gene silencing ability of an oligonucleotide or an RNA-induced silencing complex formed by the oligonucleotide can be to first synthesize the above-mentioned uracil derivative and other nucleotides, and then use the uracil derivative as a raw material to connect to the 5' end of the oligonucleotide chain through a reaction, and the 3-position oxygen atom of the uracil derivative is connected to the adjacent nucleotide residue or nucleoside residue, for example, by a solid-phase synthesis method;.

[0065] First, the present application provides an oligonucleotide, the 5' end of which is the above-mentioned uracil derivative.

[0066] Described oligonucleotide refers to the polymer or oligomer containing nucleotide residues. In the present application, described oligonucleotide can comprise single-stranded oligonucleotide and double-stranded oligonucleotide.In double-stranded oligonucleotide, the base length of each oligonucleotide chain may be different.In addition, double-stranded oligonucleotide can comprise one or more mismatched base pairs.

[0067] The 5'-terminus of the oligonucleotide is a uracil nucleotide residue whose base has been modified with the aforementioned thiophene, pyridine, or phenylpropionic acid. The base at the 5'-terminus of the oligonucleotide refers to the uracil nucleotide residue at the 5'-terminus of the oligonucleotide. In the present application, the uracil nucleotide residue at the 5'-terminus of the oligonucleotide is modified with the aforementioned thiophene, pyridine, or phenylpropionic acid groups to impart these groups to the base of the nucleotide residue.

[0068] In a specific embodiment of the present application, the 5' end of the oligonucleotide is a modified uracil nucleotide residue, and the structural formula of the natural uracil nucleotide is shown in the following formula XIII:

[0069]

[0070] In a specific embodiment of the present application, the 5' end of the oligonucleotide has a structure shown in any one of the following formulas VII to XII:

[0071]

[0072]

[0073]

[0074] wherein R1 is selected from OH or OMe.

[0075] In a preferred embodiment of the present application, the R1 is OMe, which can improve the stability of the oligonucleotide.

[0076] In a specific embodiment of the present application, the oligonucleotide may be siRNA, and the siRNA includes a sense strand and an antisense strand.

[0077] In a preferred embodiment of the present application, the oligonucleotide can be the antisense strand of siRNA. The present application performs thiophene modification, pyridine modification, or phenylpropionic acid modification on the base of the uracil nucleotide residue at the 5' end of the antisense strand of siRNA, thereby improving the ability of the antisense strand of siRNA to recognize, bind to, and silence mRNA.

[0078] The present application also provides an oligonucleotide conjugate, which contains the above-mentioned oligonucleotide and a conjugation group connected to the oligonucleotide.

[0079] The oligonucleotide conjugate refers to a compound formed by covalently linking an oligonucleotide with a chemical moiety having a specific function (such as a targeting molecule, a drug carrier, a fluorescent marker, etc.) by a chemical method.

[0080] The conjugated group may include a linker and a pharmaceutically acceptable targeting group, and may also include a delivery auxiliary group. The oligonucleotide, the linker, the targeting group, and the delivery auxiliary group are connected in sequence, and the connection may be a covalent or non-covalent connection. The targeting group is selected from a ligand that can bind to a cell surface receptor, and the delivery auxiliary group is selected from a group that can increase the biocompatibility of the oligonucleotide conjugate in the target organ or tissue for delivery.

[0081] The present application does not limit the number of the targeting groups, for example, it can be 1-6.

[0082] The conjugation site of the conjugated group to the oligonucleotide can be at the 3' end or 5' end of the sense strand of the oligonucleotide, or at the 5' end of the antisense strand of the oligonucleotide, or in the internal sequence of the oligonucleotide. For example, the conjugated group can be connected to the phosphate group, ribose sugar ring, base, 2'-hydroxyl group, or 3'-hydroxyl group of the nucleotide.

[0083] The targeting group can be a ligand that can bind to the asialoglycoprotein receptor on the surface of mammalian liver cells.

[0084] The targeting group may include, but is not limited to, N-acetylgalactosamine, mannose, arabinose, galactose, glucose, fructose, fucose, N-acetylglucosamine, sialic acid, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, and derivatives thereof.

[0085] In a specific embodiment of the present application, the oligonucleotide conjugate can be prepared by conventional methods, such as chemical synthesis, solid phase synthesis, enzymatic synthesis, and Diels-Alder reaction.

[0086] The present application also provides a pharmaceutical composition comprising an effective amount of the aforementioned oligonucleotide or the aforementioned oligonucleotide conjugate.

[0087] In this application, the effective amount refers to the amount of compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 0.01-99.99% by weight of the oligonucleotide / agent or oligonucleotide conjugate / agent of the present application; preferably, it contains 0.1-99.9% by weight of the oligonucleotide / agent or oligonucleotide conjugate / agent of the present application. More preferably, the term "one dose" refers to a complete drug dose, i.e., the amount administered in one administration, including the amount of the drug for various routes of administration, such as oral, parenteral, and topical.

[0088] The present application also provides the use of the above-mentioned uracil derivative, or the above-mentioned oligonucleotide, or the above-mentioned oligonucleotide conjugate, or the above-mentioned pharmaceutical composition in the preparation of nucleic acid drugs or target protein expression inhibitors.

[0089] In the above-mentioned use, the uracil derivatives, oligonucleotides, oligonucleotide conjugates, and pharmaceutical compositions can be used as active ingredients in the preparation of nucleic acid drugs or target protein expression inhibitors, or the uracil derivatives, oligonucleotides, oligonucleotide conjugates, and pharmaceutical compositions can be used as raw materials or reaction intermediates to prepare nucleic acid drugs or target protein expression inhibitors.

[0090] Nucleic acid drugs refer to gene therapy drugs based on DNA, RNA, or synthetic oligonucleotide analogs that exert pharmacological effects by regulating gene transcription and expression. Examples include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), aptamers, messenger RNA (mRNA), and microRNA (miRNA). In a specific embodiment of the present application, the nucleic acid drug is an siRNA drug.

[0091] In a specific embodiment of the present application, the nucleic acid drug is a drug for a disease related to the mRNA level expressed by the target gene.

[0092] Diseases associated with the mRNA level of target gene expression refer to those diseases caused by abnormal gene expression regulation (such as problems in mRNA synthesis, processing, stability and translation efficiency), so that the mRNA level of target gene expression associated with the disease can be regulated by nucleic acid drugs, including but not limited to cancer (such as breast cancer, lung cancer, colorectal cancer, prostate cancer, leukemia, lymphoma, melanoma), vascular diseases (such as myocardial infarction, heart failure, atherosclerosis, hypertension), central nervous system diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis), metabolic diseases (such as diabetes (type 1 and type 2), obesity, metabolic syndrome), autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis), inflammatory bowel disease (Crohn's disease and ulcerative colitis), Inflammation), genetic diseases (cystic fibrosis, Duchenne muscular dystrophy, sickle cell anemia, thalassemia), infectious diseases (such as HIV / AIDS, tuberculosis, hepatitis B, hepatitis C), respiratory diseases (such as asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis), liver diseases (such as non-alcoholic fatty liver disease (NAFLD), cirrhosis, liver cancer), kidney diseases (such as chronic kidney disease, acute kidney injury, diabetic nephropathy), eye diseases (such as age-related macular degeneration (AMD), glaucoma, diabetic retinopathy), bone diseases (such as osteoporosis, osteoarthritis, rheumatoid arthritis), mental illnesses (such as depression, schizophrenia, bipolar disorder), blood diseases (such as hemophilia, thrombocytopenia, aplastic anemia), skin diseases (such as psoriasis, eczema, skin cancer)

[0093] In certain specific embodiments of the present application, the nucleic acid drug is a drug for liver disease, central nervous system disease, ophthalmic disease, kidney disease, respiratory system disease, etc.

[0094] The target protein expression inhibitor refers to a compound that can specifically reduce the expression level of the target protein or inhibit its function. These inhibitors can be small molecule drugs, antibodies, nucleic acid drugs (such as siRNA, ASO) or protein-based chimeras (such as PROTAC)

[0095] The drugs or pharmaceutical compositions mentioned in the present application may also include pharmaceutically acceptable excipients or carriers. "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the mutual blending of the components in the composition without significantly reducing the efficacy of the compound. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0096] There is no particular limitation on the administration of the drug or pharmaceutical composition of the present application. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration, or subcutaneous administration).

[0097] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient of the drug is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizing agents, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0098] Solid dosage forms such as tablets, pills, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active ingredient in the drug can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If desired, the active ingredient of the drug can also be microencapsulated with one or more of the above-mentioned excipients.

[0099] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient of the drug, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0100] Besides these inert diluents, the medicament (pharmaceutical composition) may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfumes.

[0101] In addition to the active ingredient of the drug or pharmaceutical composition, the suspension may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0102] Medicaments or pharmaceutical compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0103] The target of the drug or pharmaceutical composition can specifically be various mammals, including but not limited to rodents, artiodactyls, perissodactyls, lagomorphs, primates, such as humans, monkeys, other primates, mianyang, cattle, horses, donkeys, pigs, dogs, cats, mice, rabbits, rats, guinea pigs, hamsters, foxes, deer, etc.; in a preferred embodiment of the present application, the target of the drug or pharmaceutical composition is human or mouse. When the drug (pharmaceutical composition) is used to prevent and / or treat a disease related to the mRNA level of the target gene expression in the target, the drug (pharmaceutical composition) must be accurately administered to the target at an effective dose, so that the disease originally suffered by the target can be effectively inhibited, reduced or even alleviated.

[0104] The present application also provides a method for regulating the expression level of a target gene in a cell in vitro, comprising administering an effective amount of the aforementioned oligonucleotide, or the aforementioned oligonucleotide conjugate, or the aforementioned pharmaceutical composition to the cell. The regulation may be a reduction.

[0105] The method is for non-disease diagnosis and treatment purposes, for example, the method can be used in cell culture, biopharmaceuticals, biofuels, crop improvement, etc.

[0106] In the present application, the target gene can be any gene whose expression level can be regulated by RNA interference technology, for example, including but not limited to CDK1, Cyclin B1, p21, p27, RB1, Bcl-2, Bax, Caspase-3, Caspase-8, Caspase-9, AKT1, PI3K, ERK1, ERK2, mTOR, p53, MYC, KRAS, BRAF, PTEN, GLUT1, HK2, LDHA, FASN, ACC1, NF-κB, STAT3, TNF-α, IL-6, IL-1β, VEGF, VEGFR2, HIF-1α, Angiotensin II receptor 2 (A333), ... poietin-1, Tie2, ANGPTL3, BDNF, NGF, Synapsin-1, PSD-95, Tau, DNMT1, HDAC1, EZH2, SUV39H1, BRD4, Actin, Tubulin, RhoA, Rac1, Cdc42, BRCA1, BRCA2, ATM, PARP1, XRCC1, OCT4, SOX2, NANOG, KLF4, c-Myc, HIV-1Tat, HBV X, HCV Core, HPV E6, EBV LMP1, HSP90, HIF-1α, SIRT1, FOXO1, Notch1.

[0107] The present application also provides a method for preventing, and / or alleviating, and / or treating a disease related to the mRNA level expressed by a target gene, comprising administering an effective amount of the above-mentioned oligonucleotide, or the above-mentioned oligonucleotide conjugate, or the above-mentioned pharmaceutical composition to a subject, wherein the disease related to the mRNA level expressed by the target gene is the same as described above.

[0108] The present application is described below by way of specific examples. Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all employ conventional techniques in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc. Unless otherwise specified, the instruments, reagents, and materials used in the examples can be obtained through conventional channels.

[0109] Abbreviations used in this application:

[0110] MeOH: methanol; DIEA: N,N-diisopropylethylamine; DMF: dimethylformamide; AcOH: acetic acid; DMA: dimethylacetamide; DCE: 1,2-dichloroethane; POCl3: phosphorus oxychloride; ACN: acetonitrile; EtOH: ethanol; THF: tetrahydrofuran; MsCl: methanesulfonyl chloride; Dppf: bis(diphenylphosphino)ferrocene; Et3N(TEA): triethylamine; TFA: trifluoroacetic acid; K3PO4: potassium phosphate; HATU: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DMP: Dess-Martin periodinane; DMF: N,N-dimethylformamide; UMP: uracil nucleotide

[0111] Example 1 Synthesis of Compounds 003 and 003m

[0112] The synthetic route is as follows: Compound 1 (5 g) and Compound 2 (1.2 equivalents) were dissolved in 200 mL of acetonitrile. Two equivalents of trimethylsilyl trifluoromethanesulfonate (TMSOTf) were added, and the mixture was stirred at 40°C for 180 minutes. After cooling to room temperature, the reaction solution was purified on a silica gel column to afford Compound 3 (11.2 g, 93% yield).

[0113] Compound 3 (10 g) was dissolved in 200 mL of ammonia-methanol solution and stirred at 37°C for 1 hour to remove the benzyl ester (Bz). After the reaction solution was cooled to room temperature, it was purified on a silica gel column to obtain compound 4 (9.2 g, 91% yield). Compound 4 (3.6 g) and 1.2 equivalents of diphenyl carbonate (DphC) were dissolved in 100 mL of acetonitrile and stirred at 130°C for 75 minutes to remove the 2-hydroxyl group of the ribose, obtaining compound 5 (2.6 g, 72% yield). Compound 5 (2 g) was dissolved in 50 mL of a solution containing 6-10 wt.% methoxymagnesium and stirred at 70°C for 3 days. After the reaction solution was cooled to room temperature, it was purified on a silica gel column to obtain compound 6 (1.6 g, 79% yield), which was designated as 003m. Compound 4 (1 g) was reacted with phosphorus oxychloride (POCl3) in trimethyl phosphate (TMP) at 5°C overnight. Compound 7 (0.9 g, 90% yield) was obtained by silica gel column purification and designated as 003. Compound 7 was further characterized by 15N-HSQC analysis and crystallization. Compound 6 was used to synthesize the 5' end of the antisense strand of siRNA.

[0114]

[0115] Compound 003: H-NMR (D2O, 400 MHz) δ: 7.22 (d, J = 6 Hz, 1H), 7.18 (d, J = 5.6 Hz, 1H), 6.14 (d, J = 5.6 Hz, 1H), 4.68-4.65 (m, 1H), 4.39-4.37 (m, 1H), 4.22-4.21 (m, 1H), 4.16-4.14 (m, 2H).

[0116] Example 2 Synthesis of Compounds 001 and 001m

[0117] In this example, the following compounds 1 and 2 were reacted according to Example 1 to obtain compounds 6 (001m) and 7 (001). Compound 7 was further characterized by 15N-HSQC analysis and crystallization. Compound 6 was used to synthesize the 5' end of the siRNA antisense strand.

[0118]

[0119] Compound 001: H-NMR (D2O, 400 MHz) δ: 7.86 (d, J = 5.6 Hz, 1H), 7.36 (d, J = 5.6 Hz, 1H), 6.01 (d, J = 6.4 Hz, 1H), 4.49-4.46 (m, 1H), 4.27-4.24 (m, 1H), 3.98-3.98 (m, 1H), 3.91-3.89 (m, 2H).

[0120] Example 3 Synthesis of Compounds 002 and 002m

[0121] In this example, the following compounds 1 and 2 were reacted according to Example 1 to obtain compounds 6 (002m) and 7 (002). Compound 7 was further characterized by 15N-HSQC analysis and crystallization. Compound 6 was used to synthesize the 5' end of the siRNA antisense strand.

[0122]

[0123] Compound 002: H-NMR (D2O, 400 MHz) δ: 8.38 (d, J = 3.2 Hz, 1H), 7.57 (d, J = 3.2 Hz, 1H), 6.26 (d, J = 6.4 Hz, 1H), 4.86-4.79 (m, 1H), 4.48-4.46 (m, 1H), 4.22 (s, 1H), 4.13-4.11 (m, 2H).

[0124] Referring to the above method, 015m, 015, 017m, 017, 018m and 018 were synthesized and identified, and the synthesis was confirmed to be correct.

[0125]

[0126]

[0127]

[0128] Example 4 AGO2-MID domain affinity detection

[0129] In this example, 1H-15N heteronuclear single quantum coherence (1H-15N HSQC) NMR titration experiments were performed on the monophosphates (001, 002, 003, 015, 017, and 018) synthesized in Examples 1 to 6 to measure the binding affinity of the monophosphates synthesized in the examples with hAGO2-MID, in order to evaluate the effectiveness of these newly designed base structures. It can be observed in the migration spectra after superposition that the migration spectra are as follows: Figure 1 As shown in Figure A, some of these amino acids exhibit the same movement pattern in the spectrum; in this case, 003 appears to move the farthest, while 002 moves the closest. Subsequently, the chemical shift differences of these monophosphates were analyzed separately. For each monophosphate, a number of amino acid shift points were selected that were clearly independent of the other signals in each titration. The calculation formula was Δδ = [(ΔδH)2 + (0.2*ΔδN)2]1 / 2, where ΔδH and ΔδN are the observed chemical shift changes for 1H and 15N, respectively. Furthermore, Δδ was plotted as a function of the molar ratio (nucleotide:protein), and the multimodal data were fitted using the maximum shift and dissociation constant as adjustable parameters to determine the dissociation constant (Kd). The specific operating steps of the method for testing the affinity of the AGO2 MID domain can be carried out by methods well known to those skilled in the art or according to methods described in detail in the prior art (such as siRNA potency enhancement via chemical modifications of nucleotide bases at the 5'-end of the siRNA guide strand, Fumikazu Shinohara et al., RNA, 2021(27): 163-173).

[0130] The affinity test results are as follows Figure 1As shown in Table 1 and Figure B, the Kd value for UMP was 320 ± 69 μM. Notably, the Kd value for 003 monophosphate was 172 ± 26 μM, indicating that the specific modification nearly doubled the affinity compared to UMP. The Kd values ​​for 001 and 002 monophosphates were 250 ± 28 μM and 460 ± 112 μM, respectively, suggesting that the position of the sulfur atom on the modified uridine heterocycle may affect binding affinity. Despite having similar chemical structures, 001 and 003 showed enhanced affinity, while 002 exhibited an affinity even worse than that of normal uridine, indicating that 003 has the highest affinity for the AGO2 MID domain and is significantly higher than the natural substrate, UMP.

[0131] Table 1 AGO2 MID domain affinity

[0132] Compound number Kd(μM) UMP 315.5 001 249.6 002 459.7 003 172.4 015 248.6 017 194.8 018 178.8

[0133] Example 5 Crystal structure analysis

[0134] In this example, X-ray diffraction was performed on crystals of the 001, 002, and 003 monophosphates in complex with hAGO2-MID to analyze how the newly designed monophosphates docked into the protein's binding pocket and reflect differences in their binding affinities. The initial model used protein molecules extracted from the original hAGO2-MID complexed with UMP (PDB ID: 3LUJ).

[0135] The three models obtained from the crystal data all have credible monophosphate electron density in the core pocket region of the three symmetry units in the crystal packing, and the newly designed monophosphate model can also perfectly fit into these densities ( Figure 2 A). The docking pattern of these monophosphates in the active pocket is almost identical to that of conventional UMP, especially the positions of the phosphate and ribose in the complex ( Figure 2 B). These bases form a stacking effect with Y529, and in the case of modification, the heterocyclic modification may have a greater stacking effect on tyrosine. At the same time, the same part of uridine monophosphate as the source of these designs forms two hydrogen bonds with the backbone amino acids of the rigid ring in the active pocket of hAGO2-MID ( Figure 2 B).

[0136] Compared with UMP, the results did not observe any additional or reduced interactions between 001, 002, and 003 monophosphates and hAGO2-MID. However, examination of the peripheral water molecule network revealed that 001, 002, and 003 monophosphates may stabilize the 5' end recognition core of hAGO2 by affecting water molecules, the bridge between nucleic acids and proteins ( Figure 2A) In the uridine-derived 001, 002, and 003 monophosphate structures, the water molecule in the upper right corner of the cavity interacting with the oxygen of the base appears to be conserved and structurally unchanged; the same applies to the water molecules surrounding the phosphate and ribose groups. However, the 001, 002, and 003 monophosphates in complex with hAGO2-MID all have a unique water molecule between their thiophene ring and the phosphate group, which is absent in the classic UMP-hAGO2-MID complex structure. In the 001 and 003 monophosphate complexes, this unique water molecule interacts with the hydroxyl group of Y529, forming a molecule with a length of 1.5, 2.0, and 1.5, respectively. hydrogen bonds, which may stabilize tyrosine and enhance aromatic ring stacking ( Figure 2 A). In the 002 monophosphate complex, the water molecule forms a hydrogen bond with the sulfur atom on the thiophene ring, with a length of May negatively affect the stability of the base plane and hinder stacking execution ( Figure 2 A).

[0137] Example 6 Evaluation of the in vitro and in vivo activity of non-natural nucleoside-modified siRNA

[0138] The 001m-003m modified siRNAs were synthesized using a solid phase synthesis method well known to those skilled in the art. The sequences of the siRNAs are shown in Table 2.

[0139] Table 2 Sequences of 001m to 003m modified siRNAs (aB10-mtU01 to aB10-mtU03)

[0140]

[0141] Among them, mC, mG, mA, and mU represent monomeric nucleotides modified with 2′-O-methyl (2′-OMe); fC, fG, fA, and fU represent monomeric nucleotides modified with 2′-deoxy-2′-fluoro (2′-F); and * represents a phosphorothioate bond.

[0142] The structure of mtU01 is as follows:

[0143]

[0144] The structure of mtU02 is as follows:

[0145]

[0146] The structure of mtU03 is as follows:

[0147]

[0148] First, the differences in RISC loading ability of siRNA antisense chains modified with 001m to 003m were compared in HepG2 cells.

[0149] AGO2 protein binding to siRNA guide strands was assessed in HepG2 cells by stem-loop RT-qPCR. 10 nM siRNA was co-transfected with 10 nM internal standard siRNA using RNAiMax, and cell lysates were collected 24 hours later. RISC loading efficiencies were calculated using the Ct values ​​of the IgG control and internal standard siRNA. All AGO2 RISC loading efficiencies were normalized to UMP (mU).

[0150] The results are as follows Figure 3 As shown in A, 15 The dissociation constant data of N-HSQC were consistent. 003m had the strongest affinity with AGO2 protein, followed by 001m, while 002m had the worst affinity with AGO2, which was basically consistent with the affinity of uridine modified with methoxy at the 2-position of ribose (mU).

[0151] Then, the gene silencing ability of 001m-003m modified siRNA was compared in HepG2 cells under liposome transfection.

[0152] IC50 values ​​of siRNAs were evaluated in HepG2 cells at final concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 nM using RNAiMax transfection; IC50 values ​​were calculated by nonlinear regression fitting (inhibitor vs. normalized response) in GraphPad Prism and are in nM.

[0153] The results are as follows Figure 3 As shown in B, 15 The dissociation constant data of N-HSQC and RISC-loading data were consistent. 003m-modified siRNA had the highest activity in inhibiting ANGPTL3 mRNA, with IC50 of only 20% of mU. 001m-modified siRNA had the second highest activity, while 002m-modified siRNA had the worst gene silencing ability, which was at the same level as the activity of mU-modified siRNA.

[0154] On this basis, GalNAc groups were coupled to the 3' ends of the positive strands of mU and 003m modified siRNAs, respectively, and their gene silencing abilities against ANGPTL3 mRNA in primary cynomolgus monkey liver cells were compared under free uptake conditions.

[0155] siRNA was directly added to monkey primary hepatocytes at concentrations of 0.15, 0.5, 1.5, and 5 μg / ml (to allow for free uptake without transfection reagent) and incubated for 48 hours. Residual monkey ANGPTL3 mRNA in the cells was normalized to that in the PBS group, and cynomolgus macaque GAPDH was used as an internal control.

[0156] The results are as follows Figure 3 As shown in Figure C, the ability of 003m-modified siRNA to inhibit ANGPTL3 mRNA in monkey hepatocytes was significantly better than that of mU-modified siRNA.

[0157] Finally, the gene silencing abilities of 003m-modified siRNA and mU-modified siRNA in C57BL6 mice were compared.

[0158] GalNAc-conjugated siRNAs were synthesized using a solid-phase synthesis method well known to those skilled in the art. In vivo, the gene silencing efficacy of GalNAc-conjugated siRNAs carrying 003m and unmodified UMP was compared following a single subcutaneous injection at 0.3, 1.0, and 3.0 mg / kg. Human ANGPTL3 mRNA was expressed in the livers of mice using intravenous AAV injection. Fourteen days after viral injection, 003m- and mU-modified siRNAs were subcutaneously injected at doses of 0.3, 1.0, and 3.0 mg / kg. Five days after administration, residual human ANGPTL3 mRNA in the livers was measured by qPCR and normalized to that of the PBS group. Mouse gapdh was used as an internal control.

[0159] The results are as follows Figure 3 As shown in D, it was found that at doses of 0.3, 1.0 and 3.0 mg / kg, the in vivo gene silencing ability of 003m-modified siRNA was significantly higher than that of mU-modified siRNA.

[0160] Example 7 Evaluation of Cytotoxicity of Non-natural Nucleotides

[0161] GalNAc-conjugated siRNAs were primarily localized to the liver and kidneys after subcutaneous injection. In rats, up to 60% of the siRNA accumulated in the liver, while in monkeys, this proportion reached 87%. Conversely, approximately 17% of the siRNA was distributed in the kidneys of both species.

[0162] To evaluate the effects of thiophene-modified nucleotides on cell viability and mitochondrial function, this example selected four human cell lines derived from mitochondrial-rich tissues (liver, muscle, kidney, and intestine): the liver cell line HepG2, the muscle cell line SkMC, the kidney cell line Caki-1, and the intestinal cell line COLO205. Cells were cultured in glucose-containing medium and glucose-free galactose medium to induce a metabolic shift from glycolysis to oxidative phosphorylation, thereby enhancing the cells' sensitivity to mitochondrial dysfunction. Given the potential for delayed onset of mitochondrial effects due to the high initial mitochondrial abundance, this example conducted an 11-day long-term exposure experiment with four drug stimulations. Specifically, on days 0, 3, 7, and 10, UMP and thiophene-modified UMP were added to the aforementioned cell lines in glucose or galactose medium. Each nucleotide was tested at 0, 0.16, 0.8, 4, 20, 100, and 250 μM. Cytotoxicity was assessed on day 11 using a sulforhodamine B-based assay.

[0163] As shown in Table 3, most cell lines (liver, muscle, kidney, and intestine) showed similar sensitivity to exposure to thiophene-modified nucleotides in glucose and galactose media, indicating that changes in cell viability were not primarily due to mitochondrial toxicity. However, the intestinal cell line COLO205 showed a 10-fold increase in sensitivity to 003 monophosphate nucleotides in galactose medium compared to glucose medium, with a significantly lower IC50. This observation suggests that mitochondrial damage may potentially contribute to the reduced cell viability in this cell line.

[0164] Table 3 IC50 (μM) of thiophene-modified monophosphate nucleotides (001-003)

[0165]

[0166] Example 8: Risk Assessment of Genomic Insertion of Non-natural Nucleoside-Modified siRNA

[0167] Similar to common nucleoside antiviral drugs, the specially modified nucleosides and nucleotides produced after siRNA metabolism can be gradually phosphorylated at the 5-position of the ribose sugar by intracellular phosphokinase, generating specially modified nucleotide triphosphates. These nucleotide triphosphates (NTPs) can be utilized by DNA polymerase and RNA polymerase, becoming substrates for DNA replication or RNA transcription, and incorporating into genomic DNA and RNA. Therefore, in order to accurately assess the genomic insertion risk of our newly designed nucleosides, in this example, 3 mg / kg of 003m-modified siRNA was administered together with 3 mg / kg of the classic genotoxic substance aristolochic acid to the mice expressing human ANGPTL3 described in Example 6 above. Liver tissue was then collected on days 7, 14, 28, 56, and 84 after administration, and genomic DNA and total RNA from the liver were extracted. The genomic DNA and total RNA were hydrolyzed into single nucleotides using different enzymatic hydrolysis protocols. LC-MS / MS was then used to detect the content of aristolochic acid adducts dA-AL1 and 003m in the DNA. It was found that the content of dA-ALI, a positive control, in the liver showed a gradually decreasing trend from day 7 to day 84, suggesting that dA-AL1 is gradually repaired in the genomic DNA (e.g., Figure 4 A); while mtU03 was never detected in parallel (e.g. Figure 4 B). At the same time, the ability of 003m-modified siRNA to knock down the target gene human ANGPTL3 was most obvious on the 7th day. As time went on, the level of human ANGPTL3 mRNA gradually increased (e.g. Figure 4 C). The content of 003m in the total RNA of the liver was also detected and found that its overall content did not exceed 0.4 / 10 6 Nucleotides (such as Figure 4 D) is comparable to the distribution of 003m-modified siRNA in liver cells. Therefore, it is speculated that the 003m in liver cell RNA may all come from siRNA that enters the liver cell lysosomes after administration, rather than from newly generated RNA.

[0168] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed in this application shall be covered by the claims of this application.

Claims

1. A uracil derivative, which is uracil modified with an aromatic compound, wherein the aromatic compound is selected from thiophene, pyridine, or phenylpropionic acid.

2. The uracil derivative according to claim 1, characterized in that A compound having a structure represented by any one of the following formulas I to VI, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a racemate thereof: Wherein, R1 is independently selected from OH or OMe, R2 is independently selected from OH or 3. The use of the uracil derivative according to claim 1 or 2, wherein the use is selected from: 1) Serves as the 5' end of an oligonucleotide; 2) enhancing the gene silencing ability of oligonucleotides or the RNA-induced silencing complexes they form; Preferably, the oligonucleotide is siRNA; more preferably, the oligonucleotide is the antisense strand of siRNA.

4. A method for modifying an oligonucleotide, comprising using the uracil derivative according to claim 1 or 2 as the 5' end of the oligonucleotide; preferably, the oligonucleotide is siRNA; more preferably, the oligonucleotide is the antisense strand of siRNA.

5. A method for enhancing the gene silencing ability of an oligonucleotide or an RNA-induced silencing complex formed therefrom, comprising using the uracil derivative according to claim 1 or 2 as the 5' end of the oligonucleotide; preferably, the oligonucleotide is siRNA; more preferably, the oligonucleotide is the antisense strand of the siRNA. An oligonucleotide, wherein the 5' end of the oligonucleotide is the uracil derivative according to claim 1 or 2.

7. The oligonucleotide according to claim 6, characterized in that The 5' end of the oligonucleotide has a structure represented by any one of the following formulas VII to XII: wherein R1 is selected from OH or OMe; Preferably, R1 is OMe.

8. The oligonucleotide according to claim 6, characterized in that The oligonucleotide is siRNA; preferably, the oligonucleotide is the antisense strand of siRNA. 9 . An oligonucleotide conjugate, comprising the oligonucleotide according to claim 6 and a conjugation group linked to the oligonucleotide. 10 . A pharmaceutical composition comprising an effective amount of the oligonucleotide according to claim 6 , or the oligonucleotide conjugate according to claim 9 .

11. Use of the uracil derivative according to claim 1 or 2, the oligonucleotide according to any one of claims 6 to 8, the oligonucleotide conjugate according to claim 9, or the pharmaceutical composition according to claim 10 in the preparation of a nucleic acid drug or a target protein expression inhibitor; preferably, the nucleic acid drug is an siRNA drug; more preferably, the nucleic acid drug is a drug for a disease related to the mRNA level expressed by the target gene, and more preferably, the nucleic acid drug is a drug for preventing and / or treating any one or more of the following diseases: liver disease, central nervous system disease, ophthalmic disease, kidney disease, respiratory disease, cancer, vascular disease, metabolic disease, autoimmune disease, inflammatory bowel disease, genetic disease, infectious disease, respiratory disease, liver disease, kidney disease, ophthalmic disease, bone disease, mental disease, blood disease, and skin disease.

12. A method for regulating the expression level of a target gene in a cell in vitro, comprising administering to the cell an effective amount of the oligonucleotide according to any one of claims 6 to 8, or the oligonucleotide conjugate according to claim 9, or the pharmaceutical composition according to claim 10.

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