WO2025228199A1 - Double-stranded oligonucleotide inhibiting urat1 gene expression, conjugate, pharmaceutical composition and use thereof - Google Patents

Abstract

The present invention relates to the technical field of RNAi, and specifically provides a double-stranded oligonucleotide inhibiting URAT1 gene expression, a conjugate, a pharmaceutical composition and the use thereof. The provided double-stranded oligonucleotide, conjugate and pharmaceutical composition can inhibit the expression of the URAT1 gene, so as to reduce the URAT1 protein content and significantly reduce blood uric acid, thus helping to treat and/or prevent pathological conditions or diseases related to URAT1 expression, including hyperuricemia or gout.

Description

Double-stranded oligonucleotides, conjugates, pharmaceutical compositions and their uses that inhibit URAT1 gene expression

Cross-references to related applications

This disclosure claims priority to Chinese Patent Application No. 202410537237.7, filed on April 30, 2024, entitled "Oligonucleotide conjugate for inhibiting URAT1 expression and its use thereof", and to Chinese Patent Application No. 202510436069.7, filed on April 8, 2025, entitled "Double-stranded oligonucleotide, conjugate, pharmaceutical composition for inhibiting URAT1 expression and its use thereof", the entire contents of which are incorporated herein by reference.

Technical Field

This disclosure belongs to the field of RNAi technology, specifically providing a double-stranded oligonucleotide, conjugate, pharmaceutical composition, and its use for inhibiting URAT1 gene expression.

Background Technology

Urate transporter 1 (URAT1) is the main transporter protein controlling the reabsorption of urate in the kidneys and a key target in the pathogenesis of hyperuricemia. Existing drugs that treat hyperuricemia by inhibiting URAT1 typically reduce blood uric acid levels by decreasing reabsorption, thus treating hyperuricemia and gout. However, these inhibitors usually have strong hepatotoxicity and kidney damage.

RNAi is one of the more mature treatment methods currently available. Reducing the expression of the gene encoding the URAT1 protein can decrease uric acid reabsorption and promote increased uric acid excretion, thereby lowering blood uric acid levels. However, there are currently no RNAi drugs specifically targeting URAT1 on the market.

Summary of the Invention

In view of this, the present disclosure provides a double-stranded oligonucleotide, conjugate, pharmaceutical composition and use thereof for inhibiting URAT1 expression.

In a first aspect of this disclosure, a double-stranded oligonucleotide for inhibiting URAT1 gene expression is provided, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand and the antisense strand being 17-30 nucleotides in length, and the sense strand and the antisense strand being at least partially complementary; wherein, starting from the 5' end, the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 70-138, 287-296.

In a second aspect of this disclosure, this disclosure provides a conjugate comprising the double-stranded oligonucleotide described in the first aspect of this disclosure and one or more targeting ligand groups conjugated to the double-stranded oligonucleotide.

In a third aspect of this disclosure, a pharmaceutical composition is provided comprising the double-stranded oligonucleotides described in the first aspect of this disclosure and/or the conjugates described in the second aspect of this disclosure.

In a fourth aspect of this disclosure, the present disclosure provides the use of the double-stranded oligonucleotide described in the first aspect of this disclosure, and/or the conjugate described in the second aspect of this disclosure, and/or the pharmaceutical composition described in the third aspect of this disclosure in the preparation of a medicament for inhibiting URAT1 gene expression.

In a fifth aspect of this disclosure, the present disclosure provides the use of the double-stranded oligonucleotides described in the first aspect of this disclosure, and/or the conjugates described in the second aspect of this disclosure, and/or the pharmaceutical compositions described in the third aspect of this disclosure in the preparation of a medicament for treating and/or preventing pathological conditions or diseases associated with URAT1 expression.

In a sixth aspect of this disclosure, this disclosure provides a method for inhibiting URAT1 gene expression, comprising administering to a subject an effective dose of the double-stranded oligonucleotide described in the first aspect of this disclosure, and/or the conjugate described in the second aspect of this disclosure, and/or the pharmaceutical composition described in the third aspect of this disclosure.

In a seventh aspect of this disclosure, this disclosure provides a method for preventing and/or treating hyperuricemia or gout, comprising administering to a subject an effective amount of the double-stranded oligonucleotide described in the first aspect of this disclosure, and/or the conjugate described in the second aspect of this disclosure, and/or the pharmaceutical composition described in the third aspect of this disclosure.

The double-stranded oligonucleotides, conjugates, and pharmaceutical compositions disclosed herein can inhibit the expression of the URAT1 gene, reduce the URAT1 protein content, and significantly reduce blood uric acid, which can help treat and/or prevent pathological conditions or diseases related to URAT1 expression, including hyperuricemia or gout.

Detailed Implementation

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired results.

General definitions and terminology:

Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

In this disclosure, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

In this disclosure, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

In this disclosure, the term "small interfering RNA (siRNA)" refers to a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts via the RISC pathway by forming an RNA-induced silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting the translation of mRNA into amino acids and its conversion into proteins. For example, siRNA can regulate (e.g., inhibit) the expression of URAT1 in cells.

In this disclosure, the terms "sequence" and "nucleotide sequence" refer to a sequence of nucleobases or nucleotides. As used herein, "base," "nucleotide base," or "nucleobase" is a pyrimidine or purine compound that is a component of a nucleotide and includes purine bases adenine and guanine, and pyrimidine bases cytosine, thymine, and uracil. Nucleobases may be further modified. The synthesis of modified nucleobases (including phosphorous amide compounds of modified nucleobases) is known in the art.

In this disclosure, the term "double-stranded oligonucleotide" refers to a double-stranded structure formed by two oligonucleotides through partial or complete base pairing. The two oligonucleotides include a sense strand and an antisense strand, which may or may not be of the same length. As long as at least some base-pairing regions exist to form a double-stranded region, the oligonucleotide having a double-stranded structure is considered a double-stranded oligonucleotide as described in this disclosure. The nucleotides constituting the double-stranded oligonucleotide in this disclosure can be modified or unmodified nucleotides. When referring to modified nucleotides, unless otherwise specified, the modification does not specifically refer to the modified site. In addition to the modification of the nucleotides, the linking bonds between the nucleotides in the double-stranded oligonucleotide in this disclosure may also be modified. Double-stranded oligonucleotides containing modified linking bonds between nucleotides are also considered double-stranded oligonucleotides as described in this invention. Besides the nucleotide portion, the double-stranded oligonucleotide in this disclosure may also contain compounds or modifiers acceptable in the art to improve the properties of the double-stranded oligonucleotide, such as linking ligands to form conjugates.

In this disclosure, the term "antisense strand (or guide strand)" includes a region substantially complementary to a target sequence. The term "sense strand (or follower strand)" refers to an iRNA strand containing an iRNA strand substantially complementary to the antisense strand. The term "substantially complementary" means completely complementary or at least partially complementary, for example, the antisense strand being completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches may be present in the internal or terminal regions of the molecule, wherein the most tolerant mismatches are present in the terminal regions, for example, within 5, 4, 3, or 2 nucleotides at the 5'- and/or 3' ends of the iRNA. It should be noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide substantially complementary to a continuous portion of the mRNA of interest.

In this paper, the term "target sequence" refers to a continuous portion of the nucleotide sequence of the mRNA molecule that forms during transcription of the URAT1 gene, including mRNA that is a product of RNA processing as a primary transcription product. URAT1 can be intracellular, for example, in the cells of a subject.

In this disclosure, the term “complementary” when used to describe a first nucleobase or nucleotide sequence (e.g., a positive/antisense strand of an RNAi agent or a targeting mRNA) relative to a second nucleobase or nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence (forming base-pair hydrogen bonds under mammalian physiological conditions (or similar in vitro conditions)) and form a double-stranded or double-helical structure under certain standard conditions.

In this disclosure, the term "complete complementarity" means that in a hybridized nucleobase or nucleotide sequence pair, all (100%) bases of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence. "Partial complementarity" means that in a hybridized nucleobase or nucleotide sequence pair, a majority, such as at least 70% (but not all), of the bases of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

In this disclosure, the term "substantially complementary" means that in the hybridized nucleobase or nucleotide sequence pair, the vast majority, such as at least 85% or up to three nucleotide differences (but not all), of the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may comprise all or part of the first or second nucleotide sequence.

In this disclosure, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used to refer to nucleobase or nucleotide matching between the sense and antisense strands of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of the target mRNA.

Unless otherwise specified, the term "complementarity" refers to the ability of an oligonucleotide in the first sequence to hybridize with an oligonucleotide in the second sequence under certain conditions and form a double-stranded structure. "At least partially complementary" means that the two sequences can be completely complementary, or have no more than 5, 4, 3, or 2 mismatched base pairs in total, while retaining the ability to hybridize under the relevant conditions. Furthermore, in cases where two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for determining complementarity. Correspondingly, in this invention, unless otherwise specified, "mismatch" means that in the siRNA double-stranded molecule, the bases at corresponding positions are not paired in a complementary manner.

In this disclosure, the terms "nucleotide difference," "nucleotide base difference," and "nucleotide sequence difference" are used interchangeably. A nucleotide difference refers to a change in the type of bases of nucleotides at the same or corresponding positions compared to the original nucleotide sequence. For example, if a nucleotide base in the original nucleotide sequence is A, and the nucleotide base at the same or corresponding position is changed to U, C, G, or dT, dC, dG, etc., then a nucleotide sequence difference is considered to exist at that position. It should be noted that if, compared to the original nucleotide sequence, the nucleotides at the same or corresponding positions differ only in the presence or type of modification, then a nucleotide sequence difference is not considered to exist at that position.

In this disclosure, the terms "ligand" or "conjugation group" refer to an atom or group of atoms that binds to an oligonucleotide or other oligomer. Generally, a conjugation group modifies one or more properties of the compound to which it is linked, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge, and/or clearance properties. When referring to a link between two molecules, the term "link" as used herein means that the two molecules are directly or indirectly connected by a covalent bond, or that the two molecules are associated by a non-covalent bond (e.g., a hydrogen bond or an ionic bond).

In this disclosure, the terms “link” or “association” refer to the connection between two compounds or molecules, meaning that the two molecules are linked by a covalent bond or associated via a non-covalent bond (e.g., a hydrogen bond or an ionic bond). Unless otherwise stated, the terms “link” and “association” as used in this disclosure may refer to a link between a first compound and a second compound, with or without any inserted atoms or groups of atoms.

In this disclosure, a linking group is one or more atoms that connect one molecule or a portion of a molecule to another second molecule or a second portion of a molecule. A linking group can contain any number of atoms or functional groups. In some embodiments, the linking group is used solely to link two bioactive molecules.

Unless otherwise stated, any symbol used in this disclosure refers to any group or groups that may be attached thereto, which is consistent with the scope of the invention as described in this disclosure.

According to common knowledge in the art, a peptide is a compound formed by the linkage of two or more amino acids via amide bonds. Here, individual amino acids are linked in a specific sequence to form a chain. An amino acid is a compound carrying at least one amino group and at least one carboxyl group. This includes naturally occurring amino acids (amino acids that produce proteins in vivo), non-natural amino acids, or prepared amino acids that can be found in organisms.

In this disclosure, unless otherwise stated, amino acid residues are present in the L-form.

As used in this disclosure, the term "standard amino acid" refers to the following twenty amino acids: alanine, arginine, asparagine, aspartic acid (aspartate), cysteine, glutamine, glutamic acid (glutamate), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

As used in this disclosure, the term "non-standard amino acid" refers to an amino acid other than a "standard amino acid" as defined in this disclosure. "Non-standard amino acids" include, but are not limited to, N-formylmethionine, hydroxyproline, selenomethionine, isovaleine, citrulline (Cit), ornithine, α-methyl-aspartate (αMeD), α-methyl-leucine (αMeL), N-methylalanine, N-methyl-glycine (NMeG), N-methylleucine (NMeL), O-cyclohexyl-alanine (Cha), N-ethylalanine, N,N-ε-dimethyllysine (K(Me) ₂ ), methylarginine (Arg(Me)), dimethylarginine (R(Me) ₂ ), n-alkylated L-α amino acids, and other amino acid analogs or amino acid mimics that function in a manner similar to naturally occurring amino acids.

As used in this disclosure and as understood by those skilled in the art, a polyethylene glycol (PEG) unit refers to a repeating unit of the formula ( _CH₂CH₂O ). It should be understood that in the chemical structures disclosed in this disclosure, a PEG unit can be described as ( _CH₂CH₂O ), ( _OCH₂CH₂ ), or ( _CH₂OCH₂ ). It should also be understood that _{the number} representing the _number of _repeating PEG units can be placed on either side of the brackets representing the PEG unit. It should be further understood that terminal PEG units can be capped by atoms (e.g., hydrogen atoms) or some other part.

In this disclosure, the term "cyclic peptide" refers to a polypeptide chain in which two cysteine residues are linked by a disulfide bond to form a cyclic polypeptide chain. In this disclosure, the disulfide bond between the two cysteine residues in the "cyclic peptide" is... express.

In this disclosure, the 2'-OH and 2'-H of the ribose ring in the "2',2'-[halogen, halogen] disubstituted nucleotide" are both substituted with a halogen (e.g., F, Cl, Br or I, preferably F); for example, the structural formula of the 2',2'-[F,F] disubstituted nucleotide is as follows:

In this disclosure, the 2'-OH of the ribose ring in the "2',2'-[halogen, _C1-6 alkyl] disubstituted nucleotide" is substituted with a halogen, and the 2'-H of the ribose ring is substituted with a _C1-6 alkyl group; for example, the structural formula of the 2',2'-[F, _CH3 ] disubstituted nucleotide is as follows: Wherein, Base represents a nucleoside base, which is selected from A, U, G, C or T.

In this disclosure, the structural formula of the "thiophosphate nucleoside inter-bond" is as follows: The structural formula of the "dithiophosphate nucleoside inter-bond" is:

In this disclosure, the term "pharmaceutical composition" or "composition" can refer to something used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining active siRNA with liquid excipients, finely pulverized solid excipients, or both.

In this disclosure, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and/or toxicologically compatible with other components of the formulation and/or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used in this disclosure means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

In this disclosure, the term "pharmaceutically acceptable carrier or excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients that are incompatible with the siRNA of this disclosure, such as those that produce any adverse biological effects or interactions with any other component of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

In this document, the term "subject" refers to any animal being examined, studied, or treated, and is not intended to limit this disclosure to any particular type of subject. In some embodiments, humans are preferred subjects. In other embodiments, non-human animals are preferred subjects, including but not limited to mice, monkeys, ferrets, cattle, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles. In still other embodiments, cells are preferred subjects.

In this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Here, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

In this disclosure, the terms “prevention” and “avoidance” are used interchangeably to refer to methods for obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a “preventive benefit,” the conjugate, RNAi reagent, or composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

In this disclosure, the term "administration" generally refers to the introduction of a pharmaceutical preparation of this disclosure into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug may be employed. Administration may include, but is not limited to, intravenous, intra-arterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. A daily dose may be divided into one, two, or more doses in suitable forms to be administered at one, two, or more times during a period of time.

As in this disclosure, the term "regulation of gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits, is upregulated or downregulated such that the expression, level, or activity is greater or less than that observed in the absence of a regulator. For example, the term "regulation" may mean "inhibition," but the use of the word "regulation" is not limited to this definition.

In this invention, unless otherwise specified, the term "inhibition" refers to the downregulation of target gene expression due to siRNA-mediated mRNA degradation. "Downregulation" means a decrease in target gene expression level of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more compared to the absence of siRNA treatment.

In this document, the term "inhibition of URAT1 gene expression" includes any level of inhibition of the URAT1 gene, such as at least partial inhibition of URAT1 gene expression, including inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. URAT1 gene expression can be evaluated based on the level of any variable associated with URAT1 gene expression, such as the mRNA or protein level of URAT1. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as baseline level before administration, or level measured in similar subjects, cells, or samples that have never been treated or have been treated with a control (e.g., a control with only buffer or a control without active agent).

In addition to any conventional excipients, the use of any range of siRNAs incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

In this disclosure, unless otherwise stated, all proportions of reagents used are calculated on a volume ratio (v/v).

To make the objectives, technical solutions, and advantages of this application clearer, the implementation schemes of this application will be further described in detail below with reference to the embodiments.

Double-stranded oligonucleotides:

In a first aspect of this disclosure, a double-stranded oligonucleotide for inhibiting URAT1 gene expression is provided, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand and the antisense strand being 17-30 nucleotides in length, and the sense strand and the antisense strand being at least partially complementary.

In some embodiments, the antisense strand is 21-23 nucleotides long, and the sense strand is 19-21 nucleotides long. For example, the antisense strand is 21 nucleotides long, and the sense strand is 19 nucleotides long.

Starting from the 5' end, the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 70-138 and 287-296.

In some alternative embodiments, starting from the 5' end, the antisense strand comprises no more than three nucleotides different from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 70-138, 287-296.

In some alternative embodiments, starting from the 5' end, the antisense strand comprises no more than two nucleotides different from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 70-138, 287-296.

In some alternative embodiments, starting from the 5' end, the antisense strand comprises no more than one nucleotide different from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 70-138, 287-296.

In some alternative embodiments, starting from the 5' end, the antisense strand comprises nucleotides 1-19 of the nucleotide sequence shown in any of SEQ ID NO. 70-138, 287-296.

In some specific embodiments, the antisense strand is selected from any of the nucleotide sequences shown in SEQ ID NO.70-138 and 287-296.

In some alternative embodiments, the positive strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequences shown in any of SEQ ID NO. 1-69, 277-286.

In some alternative embodiments, the positive strand comprises no more than three nucleotides different from the nucleotide sequences shown in any of SEQ ID NO. 1-69, 277-286.

In some alternative embodiments, the positive strand comprises no more than two nucleotides different from the nucleotide sequences shown in any of SEQ ID NO. 1-69, 277-286.

In some alternative embodiments, the positive strand comprises no more than one nucleotide different from the nucleotide sequences shown in any of SEQ ID NO. 1-69, 277-286.

In some specific embodiments, the positive strand is selected from any of the nucleotide sequences shown in SEQ ID NO. 1-69, 277-286.

In some alternative implementations, the sense strand and the antisense strand have a mismatch of no more than 3 nucleotides.

In some alternative implementations, the sense strand and the antisense strand have a mismatch of no more than 2 nucleotides.

In some alternative implementations, the sense strand and the antisense strand have a mismatch of no more than one nucleotide.

In some specific implementations, the justice chain and the antisense chain are completely complementary.

In some alternative embodiments, the double-stranded oligonucleotide comprises the sense strand sequence shown in any one of groups RN591001-RN591079, and the 1st to 19th nucleotide sequences of the antisense strand sequence shown in any one of groups RN591001-RN591079, starting from the 5' end:

In some specific embodiments, the double-stranded oligonucleotide is selected from any one of the groups RN591070-RN591079.

In some other specific embodiments, the double-stranded oligonucleotide is selected from any one of RN591008, RN591017, RN591023, RN591047, RN591063, RN591066, RN591075, and RN591078.

In some alternative embodiments, the double-stranded oligonucleotide contains at least one modified nucleotide. In other alternative embodiments, each nucleotide in the double-stranded oligonucleotide is selected from modified nucleotides.

In some alternative embodiments, the modified nucleotide includes at least one of the following: 2'-halogenated nucleotides (e.g., 2'-F-modified nucleotides), 2'-deoxy-modified nucleotides, 2'- _OC1-6alkyl -modified nucleotides (e.g., 2'-O- _CH3- modified nucleotides), 2'-O-( _CH2 ) _n -O-Me-modified nucleotides (e.g., _2' -O- _CH2CH2 -O- _CH3- modified nucleotides), 2'-amino-modified nucleotides, nucleotides modified with 2',2'-[halogen, halogen] disubstituted or 2',2'-[halogen, _C1-6alkyl ] disubstituted nucleotides, debased nucleotides, or nucleotide-like compounds; n is selected from 1 or 2; the nucleotide-like compounds include peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid/glycerol nucleic acid (GNA), and threose nucleic acid. At least one of the following: TNA (tranquil nucleic acid) or unlocked nucleic acid (UNA).

In some alternative embodiments, the modified nucleotide includes at least one of the following: a 2'-F modified nucleotide, a 2'- _deoxy modified nucleotide, a 2'-O-CH3 modified nucleotide, a _2' -O- _CH2CH2 -O- _CH3 modified nucleotide, or a 2',2'-[F, _CH3 ] disubstituted nucleotide. In other alternative embodiments, the modified nucleotide includes at least one of the following: a 2'-F modified nucleotide, a 2'-O- _CH3 modified nucleotide, or a 2'-O- _CH2CH2 - _O - _CH3 modified nucleotide.

In some alternative embodiments, the antisense strand of the double-stranded oligonucleotide is 21 nucleotides long, the sense strand is 19 nucleotides long, and each nucleotide in the double-stranded oligonucleotide is a modified nucleotide; wherein:

In the direction from the 5' end to the 3' end, at least four nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, and each nucleotide site other than those modified with 2'-F is independently selected from nucleotides modified with 2'-O-CH ₃ or nucleotides modified with 2'-O-CH ₂ CH ₂ -O-CH ₃ ; at least three nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and each nucleotide site other than those modified with 2'-F is selected from nucleotides modified with 2'-O-CH ₃ .

In some alternative embodiments, the antisense strand of the double-stranded oligonucleotide is 21 nucleotides long, the sense strand is 19 nucleotides long, and each nucleotide in the double-stranded oligonucleotide is a modified nucleotide; wherein:

In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F; either nucleotide at position 9 or 12 is selected from nucleotides modified with 2'-F, and the other nucleotide is selected from nucleotides modified with 2'-O-CH ₃ ; the nucleotide at position 15 is selected from nucleotides modified with 2'-O-CH ₂ CH ₂ -O-CH ₃ or nucleotides modified with 2'-O-CH ₃ ; and the nucleotides at the remaining positions are nucleotides modified with 2'-O-CH ₃ or other modified nucleotides besides those mentioned above. The nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-CH ₃ or other modified nucleotides besides those mentioned above.

In some alternative embodiments, the antisense strand of the double-stranded oligonucleotide is 21 nucleotides long, the sense strand is 19 nucleotides long, and each nucleotide in the double-stranded oligonucleotide is a modified nucleotide; wherein:

In the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F; either nucleotide at position 9 or 12 is selected from nucleotides modified with 2'-F, and the other nucleotide is selected from nucleotides modified with 2'-O-CH ₃ ; the nucleotide at position 15 is selected from nucleotides modified with 2'-O-CH ₂ CH ₂ -O-CH ₃ or nucleotides modified with 2'-O-CH ₃ ; and the nucleotides at the remaining positions are nucleotides modified with 2'-O-CH _3. The nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-CH ₃ .

In some alternative embodiments, the antisense strand of the double-stranded oligonucleotide is 21 nucleotides long, the sense strand is 19 nucleotides long, and each nucleotide in the double-stranded oligonucleotide is a modified nucleotide; wherein:

Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, the nucleotide at position 15 is selected from nucleotides modified with _2' -O- _CH2CH2 -O- _CH3 or nucleotides modified with 2'-O- _CH3 , and the nucleotides at the remaining positions are nucleotides modified with 2'-O- _CH3 or other modified nucleotides besides the above modifications; the nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are nucleotides modified with _2' -O-CH3 or other modified nucleotides besides the above modifications.

In some specific embodiments, the antisense strand of the double-stranded oligonucleotide is 21 nucleotides long, the sense strand is 19 nucleotides long, and each nucleotide in the double-stranded oligonucleotide is a modified nucleotide; wherein:

Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, the nucleotide at position 15 is selected from nucleotides modified with _2' -O- _CH2CH2 -O- _CH3 , and the nucleotides at the remaining positions are nucleotides modified with 2'-O- _CH3 ; the nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O- _CH3 .

In some alternative embodiments, the double-stranded oligonucleotide contains at least one modified internucleotide bond selected from phosphate thioester internucleotide bonds, and the phosphate thioester internucleotide bond is present at at least one of the following positions 1)-5):

1) Between the first and second nucleotides, and/or between the second and third nucleotides, starting from the 5' end of the positive strand;

2) Between the first and second nucleotides of the positive strand, starting from the 3' end, and/or between the second and third nucleotides;

3) Between the first and second nucleotides, and/or between the second and third nucleotides, starting at the 5' end of the antisense strand;

4) Between the first and second nucleotides of the antisense strand, starting from the 3' end, and/or between the second and third nucleotides, and/or between the third and fourth nucleotides;

5) Between the 10th and 11th nucleotides of the antisense strand, starting from the 5' end.

In some alternative embodiments, each nucleotide in the double-stranded oligonucleotide is a modified nucleotide.

In some alternative embodiments, at least four nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, in the direction from the 5' end to the 3' end, are selected from nucleotides modified with 2'-F, and the other nucleotide sites are each independently selected from nucleotides modified with 2'-O- _CH3 or nucleotides modified with _2' -O- _CH2CH2 -O- _CH3 ; at least three nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the other nucleotide sites are selected from nucleotides modified with 2'-O- _CH3 .

In some alternative embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are selected from 2'-F modified nucleotides; one of the nucleotides at positions 9 and 12 is selected from a 2'-F modified nucleotide, and the other nucleotide is selected from a 2'-O-CH ₃ modified nucleotide; the nucleotide at position 15 is selected from a 2'-O-CH ₂ CH ₂ -O-CH ₃ modified nucleotide or a 2'-O-CH ₃ modified nucleotide; and the nucleotides at the remaining positions are 2'-O-CH ₃ modified nucleotides. The nucleotides at positions 7-10 of the sense strand are selected from 2'-F modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-CH ₃ modified nucleotides.

In some alternative embodiments, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are selected from 2'-F modified nucleotides, one of the nucleotides at positions 9 and 12 is selected from a 2'-F modified nucleotide, the other nucleotide is selected from a 2'-O- _CH3 modified nucleotide, and the remaining nucleotides are 2'-O- _CH3 modified nucleotides; the nucleotides at positions 7-10 of the sense strand are selected from 2'-F modified nucleotides, and the remaining nucleotides are selected from 2'-O- _CH3 modified nucleotides.

In some alternative embodiments, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-CH ₃ , in the direction from the 5' end to the 3'end; the nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-CH ₃ .

In some alternative embodiments, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, and the remaining nucleotides are nucleotides modified with 2'-O- _CH3 ; the nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the remaining nucleotides are selected from nucleotides modified with 2'-O- _CH3 .

In some alternative embodiments, the double-stranded oligonucleotide further comprises at least one modified nucleotide internucleotide bond (e.g., 1, 2, 3, 4, 5, 6, 7, or 8, etc.), wherein the modified nucleotide internucleotide bond includes at least one of a thiophosphate nucleotide internucleotide bond or a dithiophosphate nucleotide internucleotide bond, and at least one of the modified nucleotide internucleotide bonds is independently selected between the first and second nucleotides of the positive strand starting at the 5' end, and between the second and third nucleotides of the positive strand starting at the 5' end. Between nucleotides, between the first and second nucleotides of the sense strand starting at the 3' end, between the first and second nucleotides of the antisense strand starting at the 5' end, between the second and third nucleotides of the antisense strand starting at the 5' end, between the tenth and eleventh nucleotides of the antisense strand starting at the 5' end, between the first and second nucleotides of the antisense strand starting at the 3' end, or between the second and third nucleotides of the antisense strand starting at the 3' end.

In some alternative embodiments, the modified nucleoside inter-bond is selected from phosphate thioside inter-bonds.

In some alternative embodiments, the double-stranded oligonucleotide has at least six modified nucleoside internucleotides (e.g., phosphate thioside internucleotides), wherein the six modified nucleoside internucleotides are selected from any six positions of the following: between the first and second nucleotides of the sense strand starting from the 5' end; between the second and third nucleotides of the sense strand starting from the 5' end; between the first and second nucleotides of the sense strand starting from the 3' end; between the first and second nucleotides of the antisense strand starting from the 5' end; between the second and third nucleotides of the antisense strand starting from the 5' end; between the tenth and eleventh nucleotides of the antisense strand starting from the 5' end; between the first and second nucleotides of the antisense strand starting from the 3' end; or between the second and third nucleotides of the antisense strand starting from the 3' end.

In some specific embodiments, the double-stranded oligonucleotide contains at least six modified nucleoside internucleotides (e.g., phosphate thioside internucleotides), wherein the six sulfur-modified nucleoside internucleotides are respectively located between the first and second nucleotides of the sense strand starting from the 5' end, between the second and third nucleotides of the sense strand starting from the 5' end, between the first and second nucleotides of the antisense strand starting from the 5' end, between the second and third nucleotides of the antisense strand starting from the 5' end, between the first and second nucleotides of the antisense strand starting from the 3' end, and between the second and third nucleotides of the antisense strand starting from the 3' end.

In some alternative embodiments, the internucleotide bond between the 10th and 11th nucleotides of the antisense strand, starting at the 5' end, is selected from modified internucleotide bonds (e.g., phosphate thioester internucleotide bonds), and the internucleotide bond between the nucleotide in the sense strand that is base-complementarily paired with the 10th nucleotide of the antisense strand and the nucleotide that is base-complementarily paired with the 11th nucleotide of the antisense strand is selected from phosphate ester internucleotide bonds.

In some alternative embodiments, the double-stranded oligonucleotide contains a dithiophosphate nucleoside bond, which is located between the first and second nucleotides of the sense strand starting from the 5' end, or between the second and third nucleotides of the sense strand starting from the 5' end, or between the first and second nucleotides of the sense strand starting from the 3' end, or between the first and second nucleotides of the antisense strand starting from the 5' end, or between the second and third nucleotides of the antisense strand starting from the 5' end, or between the tenth and eleventh nucleotides of the antisense strand starting from the 5' end, or between the first and second nucleotides of the antisense strand starting from the 3' end, or between the second and third nucleotides of the antisense strand starting from the 3' end.

In some alternative embodiments, the first nucleotide of the antisense strand, starting from the 5' end, is selected from... Or a nucleotide modified with a 5'-phosphate analog; said nucleotide is selected from any nucleotide with the following structure:

Wherein, Base represents a nucleoside base, which is selected from A, U, G, C or T; R represents -H, -OH, _-CH3 , _-OCH3 , -F.

In some alternative embodiments, the 5'-phosphate analog-modified nucleotide is selected from nucleotides modified with 5'-(E)-vinylphosphonate (5'-(E)-VP).

In some alternative embodiments, the first nucleotide of the antisense strand, starting from the 5' end, is selected from... Alternatively, the first nucleotide of the antisense strand, starting from the 5' end, may be selected from a nucleotide modified with 5'-(E)-VP. In some specific embodiments, the first nucleotide of the antisense strand, starting from the 5' end, may be selected from... In some other specific embodiments, the first nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with 5'-(E)-VP.

In some alternative embodiments, starting from the 5' end, the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequences 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 208-276, 307-316.

In some alternative embodiments, starting from the 5' end, the antisense strand comprises no more than three nucleotides different from the nucleotides at positions 1-19 of the nucleotide sequences shown in any of SEQ ID NO. 208-276, 307-316.

In some alternative embodiments, starting from the 5' end, the antisense strand contains no more than two nucleotides differing from the nucleotide sequences shown in any of SEQ ID NO. 208-276, 307-316 at positions 1-19.

In some alternative embodiments, starting from the 5' end, the antisense strand contains no more than one nucleotide from positions 1 to 19 that differ from the nucleotide sequences shown in any of SEQ ID NO. 208-276, 307-316.

In some alternative embodiments, starting from the 5' end, the antisense strand comprises nucleotides 1-19 of the nucleotide sequence shown in any of SEQ ID NO. 208-276 and 307-316;

In some specific embodiments, the antisense strand is selected from any of the nucleotide sequences shown in SEQ ID NO. 208-276 and 307-316.

In some alternative embodiments, the positive strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequences shown in any of SEQ ID NO. 139-207, 297-306.

In some alternative embodiments, the positive strand comprises no more than three nucleotides different from the nucleotide sequences shown in any of SEQ ID NO. 139-207, 297-306.

In some alternative embodiments, the positive strand contains no more than two nucleotides different from the nucleotide sequences shown in any of SEQ ID NO. 139-207, 297-306.

In some alternative embodiments, the positive strand comprises no more than one nucleotide different from the nucleotide sequences shown in any of SEQ ID NO. 139-207, 297-306.

In some specific embodiments, the positive strand is selected from any of the nucleotide sequences shown in SEQ ID NO. 139-207 and 297-306.

In some alternative embodiments, the double-stranded oligonucleotide is selected from the sense strand sequence shown in any one of groups RX591070-RX591079, and the 1st to 19th nucleotide sequences of the antisense strand sequence shown in any one of groups RX591070-RX591079, starting from the 5' end:

In some specific embodiments, the double-stranded oligonucleotide is selected from any one of RX591001-RX591079.

In some other specific embodiments, the double-stranded oligonucleotide is selected from any one of RX591008, RX591017, RX591023, RX591047, RX591063, RX591066, RX591075, and RX591078.

Conjugates:

In a second aspect, this disclosure provides a conjugate comprising the double-stranded oligonucleotide described in the first aspect of this disclosure and one or more targeting ligand groups conjugated to the double-stranded oligonucleotide.

In some alternative embodiments, the targeting ligand group targets the renal megalin receptor or the renal natriuretic peptide receptor.

In some alternative embodiments, the targeting ligand group is selected from polypeptide fragments containing at least four amino acids.

In some alternative embodiments, the targeting ligand group comprises any of the polypeptide fragments shown in 1)-6) below:

1) _-X1 (KKEEE) _n - _Km- , where n＝1-5, m＝0 or 1 or 2, and _X1 is selected from any L-α amino acid or L-β amino acid other than K and E;

2)-CLPVASC-, preferably in cyclic peptide form

3)-CYFQNC-, preferably in cyclic peptide form

4)-KIDRI-;

5)-IDRI-(Ile-Asp-Arg-Ile-);

6)-dXa-Ser-dXb-X _2- dXc-Gly-Xd-Ile-Asp-Arg(Ak)-Ile-; where X ₂ is selected from any non-natural amino acid; dXa, dXb, dXc are selected from any D-type amino acid; Arg(Ak) is selected from arginine or alkylated arginine; Xd is selected from Hyp or Pro.

In some alternative embodiments, the targeting ligand group is selected from polypeptides comprising any of the amino acid sequence segments shown in a)-g).

a)-KKEEE-KKEEE-KKEEE-K-；

b)-CKKEEE-KKEEE-KKEEE-K；

c)-CLPVASC-, preferably in cyclic peptide form

d)-KIDRI-;

e)-CYFQNCPRG-, preferably in cyclic peptide form

f)-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-;

g)-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-.

In some alternative embodiments, the targeting ligand group is linked to the double-stranded oligonucleotide via a linker;

Optionally, the structural unit of the targeting ligand group-connector in the conjugate is selected from any of the structures shown in 1)-7) below:

1)La-KKEEE-KKEEE-KKEEE-K-Laa;

2)La-CKKEEE-KKEEE-KKEEE-K-Laa;

3) La-CLPVASC-Laa, preferably in cyclic peptide form.

4)La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Laa;

5) La-CYFQNCPRG-Laa; preferably cyclic peptide form.

6)La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Laa;

7) Lb-KIDRI-Laa;

In formulas 1)-6), either La or Laa is a linker and the other is a terminal blocking group;

In formula 7), either Lb or Laa is a linker, and the other is a terminal blocking group;

The connector is a linker group containing at least one of the following groups: triazole group, PEG unit, or acyl group.

In some alternative embodiments, in formulas 1)-6), La is a linker, Laa is a terminal blocking group, and La is selected from linking groups including _{-PEG2- CH2CH2CO-} or _-triazolyl - _PEG2- ; Laa is selected from amino or alkyl-substituted amino groups, such as _-NHCH3 .

In some alternative embodiments, in Formula 7), Lb is a linker and Laa is a terminal closing group. Lb is selected from a linker group containing a 6-10 membered aromatic ring or a heteroaromatic ring; Laa is selected from an amino or alkyl-substituted amino group, such as _-NHCH3 .

In some alternative embodiments, the targeting ligand group is connected via a linker to the ends of the sense and/or antisense strands of the double-stranded oligonucleotide (e.g., the 3' end of the sense strand, and/or the 5' end of the sense strand, and/or the 3' end of the sense strand).

In some alternative embodiments, the structure of the targeting ligand group-connector in the conjugate is selected from any of the structures shown in a)-j), or a pharmaceutically acceptable salt thereof:

a)-La-KKEEE-KKEEE-KKEEE-K-Lc;

b)-La-CKKEEE-KKEEE-KKEEE-K-Lc;

c)-La-CLPVASC-Lc; preferably cyclic peptide form

d)-La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Lc;

e)-La-CYFQNCPRG-Lc; preferably in cyclic peptide form.

f)-La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Lc;

g)

h)

i)

j)

Wherein, La is a linker, which is independently selected from any bond, -NH-, amide group or linking group containing at least one PEG unit; Lc is a terminal blocking group.

In some alternative embodiments, La is a linking group containing at least two PEG units.

In some alternative embodiments, La is selected from amide groups, or n is 1-3 (e.g., 1, 2, 3), m is 0 or 1, and Z is -CO- (carbonyl) or -NH-. * represents a site covalently linked to a double-stranded oligonucleotide, and * represents a site covalently linked to a polypeptide fragment.

In some alternative embodiments, Lc is a blocking group at the C-terminus of the polypeptide fragment. In other alternative embodiments, the Lc is selected from amino or alkyl-substituted amino groups (e.g., _-NHCH3 ).

Pharmaceutical composition:

In a third aspect of this disclosure, a pharmaceutical composition is provided comprising the double-stranded oligonucleotide described in the first aspect of this disclosure and/or the conjugate described in the second aspect of this disclosure.

In some alternative embodiments, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers or excipients.

Uses in the preparation of drugs that inhibit URAT1 gene expression:

In a fourth aspect of this disclosure, the present disclosure provides the use of the double-stranded oligonucleotides described in the first aspect of this disclosure, and/or the conjugates described in the second aspect of this disclosure, and/or the pharmaceutical compositions described in the third aspect of this disclosure in the preparation of a medicament for inhibiting URAT1 gene expression.

Use in the preparation of medicaments for the treatment and/or prevention of pathological conditions or diseases associated with URAT1 expression:

In a fifth aspect of this disclosure, this disclosure provides the use of the double-stranded oligonucleotides described in the first aspect of this disclosure, and/or the conjugates described in the second aspect of this disclosure, and/or the pharmaceutical compositions described in the third aspect of this disclosure, in a medicament for treating and/or preventing pathological conditions or diseases associated with URAT1 expression.

In some alternative implementations, the pathological condition or disease is hyperuricemia or gout.

Methods to suppress URAT1 gene expression:

In a sixth aspect of this disclosure, this disclosure provides a method for inhibiting URAT1 gene expression, comprising administering to a subject an effective dose of the double-stranded oligonucleotide of the first aspect of this disclosure, and/or the conjugate of the second aspect of this disclosure, and/or the pharmaceutical composition of the third aspect of this disclosure.

In some alternative embodiments, the subject is a human being. In other alternative embodiments, the subject is a cell, such as Vero cells or HuH1 cells.

Methods for preventing and/or treating hyperuricemia or gout:

In a seventh aspect of this disclosure, this disclosure provides a method for preventing and/or treating hyperuricemia or gout, comprising administering to a subject an effective dose of the double-stranded oligonucleotide of the first aspect of this disclosure, and/or the conjugate of the second aspect of this disclosure, and/or the pharmaceutical composition of the third aspect of this disclosure.

In some alternative implementations, the subject is a human being.

The effective amount of the double-stranded oligonucleotides, double-stranded oligonucleotide conjugates, or pharmaceutical compositions described in this disclosure may vary depending on the administration method and the severity of the disease to be treated. Specifically, the effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

The medication may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral and sublingual administration), such as intravenous administration.

The double-stranded oligonucleotides, double-stranded oligonucleotide conjugates, and pharmaceutical compositions disclosed herein can effectively inhibit URAT1 gene expression, which can help treat and/or prevent pathological conditions or diseases related to URAT1 expression.

To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below with reference to examples.

Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available. The main reagents and consumables are shown in Table 1, and the main instruments and equipment are shown in Table 2.

Table 1 Main Reagents and Consumables

Table 2 Main Instruments and Equipment

Preparation Example 1: Synthesis of siRNA

siRNA sequence design:

The disclosed siRNA sequences target all transcripts and predicted transcripts of the URAT1 gene (NM_144585.4, NM_153378.3, NM_001276326.2, NM_001276327.2, XM_006718430.5, XM_006718431.5, XM_054367604.1, XM_054367605.1). The URAT1 gene sequence information is obtained from the NCBI gene database (https://www.ncbi.nlm.nih.gov/gene/).

siRNA synthesis:

(1-1) Composition of the Justice Chain (SS) and Antisense Chain (AS):

The phosphoramidol solid-phase synthesis method for nucleic acids involves starting a cycle using the aforementioned compound (e.g., CPG carrier, PS carrier) linked to a solid-phase support, and then linking nucleoside monomers one by one in the direction from the 3' end to the 5' end of the nucleotide sequence.

Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below:

The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, and the deprotection reagent was a 3% (v/v) dichloroacetic acid solution in dichloromethane. The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

The conditions for each coupling reaction were identical. The coupling reaction conditions were as follows: temperature 25℃, molar ratio of nucleic acid sequence to nucleoside monomer on the solid-phase support 1:10, molar ratio of nucleic acid sequence to coupling reagent on the solid-phase support 1:65, reaction time 600 seconds, coupling reagent 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole, and thioreagent 0.2mol/L acetonitrile/pyridine mixed solution of hydrogenated xanthanin (acetonitrile and pyridine volume ratio 1:1).

The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; and the capping reagent solution was a mixture of Cap1 and Cap2. Cap1 was a 20% (v/v) N-methylimidazole pyridine/acetonitrile mixture, with a volume ratio of pyridine to acetonitrile of 3:5. Cap2 was a 20% (v/v) acetic anhydride acetonitrile solution. The molar ratio of N-methylimidazole in Cap1 and acetic anhydride in Cap2 to the nucleic acid sequence linked on the solid-phase support was 1:1:1.

The conditions for each oxidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution, with a molar ratio of iodine to the nucleic acid sequence linked on the solid support in the coupling reaction of 30:1; the oxidation reaction was carried out in a water/pyridine mixed solvent (water to pyridine volume ratio 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution, with a molar ratio of thioreagent to the nucleic acid sequence linked on the solid support in the coupling reaction of 4:1; the thioreagent reaction was carried out in a water/pyridine mixed solvent (water to pyridine volume ratio 1:9).

After the last nucleoside monomer was ligated, the nucleic acid sequence ligated on the solid-phase support was sequentially cut, deprotected, purified, and desalted, and then freeze-dried to obtain the positive strand, wherein:

The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25 wt% ammonia solution at a concentration of 0.5 mL/μmol. The reaction was carried out at 55 °C for 16 hours. The solvent was removed, and the solution was concentrated to dryness under vacuum. After ammonia treatment, the product was dissolved in 0.4 mL/μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 mL/μmol triethylamine and 0.6 mL/μmol triethylamine trifluoride were added to deprotect the 2'-O-TBDMS protection on the ribose.

Purification and desalting conditions: Nucleic acid purification was performed using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically: eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water/acetonitrile mixture (water to acetonitrile volume ratio of 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water/acetonitrile mixture (water to acetonitrile volume ratio of 9:1); the elution gradient was eluent 1: eluent 2 = (100:0) - (50:50). The product eluates were collected and combined, and desalting was performed using a reverse chromatographic purification column. Desalting conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value and the theoretical value were consistent, it indicated that the target sense and antisense strands were obtained.

(1-2) Synthesis of siRNA:

The sense and antisense strands synthesized in step (1-1) were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture was then slowly cooled to room temperature and kept at room temperature for 10 minutes to allow the sense and antisense strands to form a double-stranded structure through hydrogen bonds, thereby obtaining the siRNA shown in Tables 4a and 4b.

Table 3a. Nucleotide sequence information of unmodified siRNA:

Table 4a. Modified siRNA sequence information:

Table 3b. Unmodified siRNA sequence information:

Table 4b. Modified siRNA sequence information:

Unless otherwise specified, the base composition and modifications described in this disclosure have the following meanings: uppercase letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide represented by the uppercase letter to the left of m is modified with 2'-O-CH ₃ ; lowercase letter f indicates that the nucleotide represented by the uppercase letter to the left of f is modified with 2'-F; (moe) indicates that the nucleotide represented by the uppercase letter to the left of (moe) is modified with 2'-O-CH ₂ CH ₂ -O-CH ₃ ; lowercase letter s indicates that the nucleoside bond between the two nucleotides to the left and right of s is a phosphate thioester nucleoside bond.

The structural formula of the nucleotide modified with 2'-O- _CH3 is as follows:

The structural formula of the nucleotide modified by 2'-F is:

The structural formula of the nucleotide modified with 2'-O- _CH2CH2 - _O - _CH3 is as follows:

Wherein, Base represents a nucleoside base, which is selected from A, U, G, C, or T. Q represents -OH, ^-O- , -SH, or ^-S- .

Unless otherwise stated, all siRNA sequences (modifications) used in this disclosure were synthesized by Suzhou Xuanjing Biotechnology Co., Ltd.

Biological testing experiments

Unless otherwise stated, all sequences used in biological detection experiments in this disclosure are modified siRNAs.

Unless otherwise stated, all PCR primers used in this disclosure were synthesized by Beijing Qingke Biotechnology Co., Ltd.

Unless otherwise stated, in real-time quantitative PCR, the ΔΔCt method is used to calculate the relative quantitative levels and inhibition rates of target gene mRNA in each test group. The calculation method is as follows:
ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group)
ΔCt(blank control group) = Ct(target gene in blank control group) - Ct(internal reference gene in blank control group)
ΔCt(test group) = ΔCt(test group) - ΔCt(mean of blank control group)
ΔCt(blank control group) = ΔCt(blank control group) - ΔCt(blank control group average)

In cell experiments, ΔCt (mean of the blank control group) is the arithmetic mean of the ΔCt values of several culture wells in the blank control group. Therefore, each sample in both the test group and the blank control group corresponds to a ΔCt value.
The relative expression level of the target gene mRNA in the test group = 2 ^{- ΔΔCt(test group)} × 100%

Using the blank control group as a baseline, the relative expression level of the target gene mRNA in the test group was normalized, and the relative expression level of the target gene mRNA in the blank control group was defined as 100%.

The inhibition rate of target gene mRNA expression in the test group = 100% - the relative expression level of target gene mRNA in the test group

Unless otherwise stated, all data on inhibitory activity are presented in accordance with the experimental figures. (X±STDEV) indicates that all experimental data were plotted and analyzed using GraphPad Prism 8.0 software.

Example 1: Evaluation of the inhibitory activity of siRNA sequences on the target gene urate transporter 1 (URAT1) in Vero cells :

In this embodiment, an in vitro cell transfection method was used to evaluate the inhibitory activity of siRNA sequences on the target gene URAT1 in Vero cells.

Vero cells (gifted by Beijing Institute of Technology) were cultured in complete medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics) at 5% _CO2 and 37°C. After reaching 80% confluence, the cells were digested with trypsin and resuspended. The resuspended cells were then adjusted to a density of 0.6 × ^{10⁵ cells} /well in complete medium and seeded into 24-well plates. After 24 hours of culture, transfection was performed.

Preparation of siRNA transfection mixture: Prepare a 20 μM stock solution of siRNA with PBS. Then, add 1.5 μL of the siRNA stock solution to each well and dilute it with 48.5 μL of Opti-MEM medium to prepare 50 μL of siRNA working solution. Add 3 μL of the stock solution to each well. The RNAiMAX transfection reagent was diluted in 47 μl of Opti-MEM medium and incubated at room temperature for 5 minutes to prepare a 50 μl working solution for transfection. Then, 50 μl of siRNA working solution was mixed with the 50 μl working solution of the transfection reagent to prepare the siRNA transfection mixture. For the MOCK group, 50 μl of Opti-MEM medium was mixed with 50 μl of the working solution of the transfection reagent.

On the day of transfection, replace 500 μl of Opti-MEM medium per well in each 24-well plate, and add 100 μl of the corresponding siRNA transfection mixture or MOCK group transfection mixture to each well. The final concentration of siRNA is 50 nM per well.

After culturing for another 24 hours, total RNA was extracted using a chemical extraction method. Specifically, after removing the culture medium from each well, 1 mL of Trizol solution was added, and cells were lysed by pipetting. The lysate was transferred to a 1.5 mL centrifuge tube, and 200 μL of chloroform was added. The tube was vortexed and incubated at room temperature for 3 min. The cells were then centrifuged at 12000 rpm for 10 min at 4 °C. 400 μL of the supernatant was transferred to a centrifuge tube containing 400 μL of isopropanol, mixed, and incubated at room temperature for 10 min. The tube was then centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was discarded. 1 mL of 75% ethanol was added, and the centrifuge tube was inverted to wash the precipitate. The tube was then centrifuged at 12000 rpm for 5 min at 4 °C, the supernatant was discarded, and the cells were air-dried at room temperature to obtain total RNA.

Take 1 μg of total RNA, and use a reverse transcription kit (Promega, Reverse Transcription System, A3500) with Oligo(dT)15 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the instructions and complete the reverse transcription reaction. After the reaction, add 80 μL of RNase-free water to the reverse transcription system to obtain a cDNA solution. Then, use a real-time quantitative PCR kit (ABI, SYBR ^™ Select Master Mix, Catalog number: 4472908) to detect the expression level of the target gene mRNA in Vero cells. In this real-time quantitative PCR method, primers targeting the target gene and primers targeting the internal reference gene were used to detect the target gene and the internal reference gene, respectively. Prepare a 20 μL Real-time PCR reaction system for each PCR detection well according to the instructions of the Real-time PCR kit. Each reaction system contains 5 μL of cDNA solution obtained from the above reverse transcription reaction, 10 μL of SYBR ^™ Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of RNase-Free H2O. Place the prepared reaction system on a Real-time PCR instrument (ABI, StepOnePlus ^™ ) and perform Real-time PCR amplification using a three-step method. The amplification program is as follows: 95℃ pre-denaturation for 10 min, then 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. Repeat the denaturation, annealing, and extension process for 40 cycles.

Table 5. Sequence list of primers used in Example 1

The results of Example 1 show that all designed sequences can effectively reduce the expression of URAT1 mRNA, among which RX591008, RX591011, RX591013, RX591014, RX591017, RX591018, RX591019, RX591022 and RX591024 sequences have relatively better gene repression levels (Table 6).

Table 6 shows the inhibitory activity of Vero cells on the target genes after administration of the siRNA conjugate described in this embodiment.

Example 2: Evaluation of the inhibitory activity of siRNA sequence on the target gene URAT1 in HuH1 cells:

In this embodiment, an in vitro cell transfection method was used to evaluate the inhibitory activity of siRNA sequences on the target gene URAT1 in HuH1 cells.

HuH1 cells (Wuhan Pronosai Biotechnology Co., Ltd.) were cultured in complete medium (DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics) at 5% CO2 and 37°C. Once the cell density reached 80%, the cells were digested with trypsin and resuspended. The resuspended cells were then adjusted to a density of 0.6 × 10⁵ cells/well in complete medium and seeded into 24-well plates. After 24 hours of culture, transfection was performed.

Preparation of siRNA transfection mixture: Prepare stock solutions of siRNA with PBS buffer at concentrations of 20 μM, 0.4 μM, and 0.2 μM, respectively. Then, take 1.5 μl of the siRNA stock solution from each well and dilute it with 48.5 μl of Opti-MEM medium to prepare 50 μl of siRNA working solution; add 3 μl of the stock solution to each well. The RNAiMAX transfection reagent was diluted in 47 μl of Opti-MEM medium and incubated at room temperature for 5 minutes to prepare a 50 μl working solution for transfection. Then, 50 μl of siRNA working solution was mixed with the 50 μl working solution of the transfection reagent to prepare the siRNA transfection mixture. For the MOCK group, 50 μl of Opti-MEM medium was mixed with 50 μl of the working solution of the transfection reagent.

On the day of transfection, replace 500 μl of Opti-MEM medium per well in each 24-well plate, and add 100 μl of the corresponding siRNA transfection mixture or MOCK group transfection mixture to each well. The final siRNA concentrations are 50 nM/well, 1 nM/well, and 0.5 nM/well, respectively.

After culturing for another 24 hours, total RNA was extracted using a chemical extraction method. Specifically, after removing the culture medium from each well, 1 mL of Trizol solution was added, and cells were lysed by pipetting. The lysate was transferred to a 1.5 mL centrifuge tube, and 200 μL of chloroform was added. The tube was vortexed and incubated at room temperature for 3 min. The cells were then centrifuged at 12000 rpm for 10 min at 4 °C. 400 μL of the supernatant was transferred to a centrifuge tube containing 400 μL of isopropanol, mixed, and incubated at room temperature for 10 min. The tube was then centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was discarded. 1 mL of 75% ethanol was added, and the centrifuge tube was inverted to wash the precipitate. The tube was then centrifuged at 12000 rpm for 5 min at 4 °C, the supernatant was discarded, and the cells were air-dried at room temperature to obtain total RNA.

To detect the mRNA expression level of the target gene: Take the total RNA (1 μg) mentioned above and perform real-time quantitative PCR according to the method described in the TaqMan Fast Advanced Master Mix (ABI, Catalog number: 4444557) manual. That is, each reaction well contains 20 μL of real-time PCR reaction system, and each reaction system contains 5 μL of cDNA solution obtained from the above reverse transcription reaction, 10 μL of TaqMan Fast Advanced Master Mix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 0.2 μL of 10 μM probe, and 3 μL of RNase-free _H2O . The prepared reaction system was placed on a real-time quantitative PCR instrument (ABI, StepOnePlus ^™ /7500) and amplified using a two-step method. The amplification program was 50℃ pre-denaturation for 2 min, 95℃ pre-denaturation for 20 s, then 95℃ denaturation for 3 s, and 60℃ annealing for 30 s. The denaturation and annealing process was repeated for 40 cycles.

Table 7 Primer sequence information used in Example 2:

The results of Example 2 (Tables 8-9) show that all designed sequences can effectively reduce the expression of URAT1 mRNA, among which the sequences RX591008, RX591017, RX591023, RX591047, RX591063, RX591066, RX591075, and RX591078 have relatively better gene repression levels.

Table 8 shows the inhibitory activity of the target gene URAT1 in HuH1 cells at a final siRNA sequence transfection concentration of 50 nM/well in Example 2:

Table 9. Inhibitory activity of siRNA sequence transfection against the target gene URAT1 in HuH1 cells at final concentrations of 1 nM/well and 0.5 nM/well in Example 2:

The above specific embodiments are merely illustrative of the present invention and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims (19)

A double-stranded oligonucleotide for inhibiting URAT1 gene expression, characterized in that: the double-stranded oligonucleotide comprises a sense strand and an antisense strand, the lengths of the sense strand and the antisense strand being 17-30 nucleotides each, and the sense strand and the antisense strand being at least partially complementary; wherein, starting from the 5' end, the antisense strand comprises at least 17 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 70-138, 287-296. According to claim 1, the double-stranded oligonucleotide is characterized in that: the positive strand comprises at least 17 consecutive nucleotides that differ from the nucleotide sequences shown in any one of SEQ ID NO. 1-69 and 277-286 by no more than 3 nucleotides; Optionally, the antisense strand is 21-23 nucleotides long; the sense strand is 19-21 nucleotides long. According to claim 1, the double-stranded oligonucleotide is characterized in that, starting from the 5' end, the antisense strand contains no more than 3 nucleotides that differ from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 70-138 and 287-296; Optionally, starting from the 5' end, the antisense strand comprises no more than two nucleotides different from the nucleotides at positions 1-19 of the nucleotide sequences shown in any of SEQ ID NO.70-138, 287-296; Optionally, starting from the 5' end, the antisense strand contains no more than one nucleotide different from the nucleotides at positions 1-19 of the nucleotide sequences shown in any of SEQ ID NO.70-138, 287-296; Optionally, starting from the 5' end, the antisense strand comprises nucleotides 1-19 of the nucleotide sequences shown in any of SEQ ID NO. 70-138 and 287-296; Optionally, the antisense strand is selected from any of the nucleotide sequences shown in SEQ ID NO.70-138 and 287-296. The double-stranded oligonucleotide according to any one of claims 1-3 is characterized in that: there is a mismatch of no more than 3 nucleotides between the sense strand and the antisense strand; Optionally, the sense strand and the antisense strand may have a mismatch of no more than 2 or 1 nucleotides; Optionally, the justice chain and the antisense chain are completely complementary. The double-stranded oligonucleotide according to any one of claims 1-4 is characterized in that: the double-stranded oligonucleotide comprises the sense strand sequence shown in any one of groups RN591001-RN591079, and the 1st to 19th nucleotide sequences of the antisense strand sequence shown in any one of groups RN591001-RN591079, starting from the 5' end:


Optionally, the double-stranded oligonucleotide is selected from any one of the groups RN591001-RN591079. The double-stranded oligonucleotide according to any one of claims 1-5 is characterized in that: the double-stranded oligonucleotide contains at least one modified nucleotide; Optionally, each nucleotide in the double-stranded oligonucleotide is selected from modified nucleotides; Optionally, the modified nucleotide includes at least one of the following: 2'-halogenated nucleotide, 2'-deoxygenated nucleotide, 2'- _OC1-6alkyl -modified nucleotide, 2'-O-( _CH2 ) _n -O-Me-modified nucleotide, 2'-amino-modified nucleotide, nucleotide modified with 2',2'-[halogen, halogen] disubstituted or 2',2'-[halogen, _C1-6alkyl ] disubstituted nucleotide, debased nucleotide, or nucleotide-like nucleotide; n is selected from 1 or 2; the nucleotide-like nucleotide includes at least one of peptide nucleic acid (PNA), morpholine nucleic acid (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), threonucleotide (TNA), or non-locked nucleic acid (UNA); Optionally, the modified nucleotide includes at least one of the following: 2'-F modified nucleotide, 2'-deoxy modified nucleotide, 2'-O-CH ₃ modified nucleotide, 2'-O-CH ₂ CH ₂ -O-CH ₃ modified nucleotide, or 2',2'-[F,CH ₃ ] disubstituted nucleotide. According to claim 6, the double-stranded oligonucleotide is characterized in that: the antisense strand of the double-stranded oligonucleotide is 21 nucleotides long, the sense strand is 19 nucleotides long, and each nucleotide in the double-stranded oligonucleotide is a modified nucleotide; wherein: In the direction from the 5' end to the 3' end, at least four nucleotides at positions 2, 6, 9, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-CH 3 or nucleotides modified with 2'-O-CH ₂ CH ₂ -O-CH ₃ ; at least three nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'- _F , and the remaining positions are nucleotides modified with 2'-O-CH ₃ . Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, the nucleotide at position 15 is selected from nucleotides modified with _2' -O- _CH2CH2 - _O -CH3, and the nucleotides at the remaining positions are nucleotides modified with 2'- _O -CH3; the nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O- _CH3 . Optionally, the double-stranded oligonucleotide contains at least one modified internucleotide bond, the modified internucleotide bond being selected from phosphate thioester internucleotide bonds, and the phosphate thioester internucleotide bond being present at at least one of the following positions 1)-5): 1) Between the first and second nucleotides, and/or between the second and third nucleotides, starting from the 5' end of the positive strand; 2) Between the first and second nucleotides of the positive strand, starting from the 3' end, and/or between the second and third nucleotides; 3) Between the first and second nucleotides, and/or between the second and third nucleotides, starting at the 5' end of the antisense strand; 4) Between the first and second nucleotides of the antisense strand, starting from the 3' end, and/or between the second and third nucleotides, and/or between the third and fourth nucleotides; 5) Between the 10th and 11th nucleotides of the antisense strand, starting from the 5' end. According to claim 6, the double-stranded oligonucleotide is characterized in that: each nucleotide in the double-stranded oligonucleotide is a modified nucleotide; wherein: In the direction from the 5' end to the 3' end, at least four nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, and the other nucleotide sites are each independently selected from nucleotides modified with 2'-O-CH ₃ or nucleotides modified with 2'-O-CH ₂ CH ₂ -O-CH ₃ ; at least three nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the other nucleotide sites are selected from nucleotides modified with 2'-O-CH ₃ . Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F; one nucleotide at position 9 and 12 is selected from nucleotides modified with 2'-F, and the other nucleotide is selected from nucleotides modified with 2'-O-CH ₃ ; the nucleotide at position 15 is selected from nucleotides modified with 2'-O-CH ₂ CH ₂ -O-CH ₃ or nucleotides modified with 2'-O-CH ₃ ; and the nucleotides at the remaining positions are nucleotides modified with 2'-O-CH _3. The nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-CH ₃ . Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-CH ₃ ; the nucleotides at positions 7-10 of the sense strand are selected from nucleotides modified with 2'-F, and the nucleotides at the remaining positions are selected from nucleotides modified with 2'-O-CH ₃ . Optionally, the double-stranded oligonucleotide further comprises at least one modified nucleoside internucleotide bond, the modified nucleoside internucleotide bond including at least one of thiophosphate nucleoside internucleotide bonds or dithiophosphate nucleoside internucleotide bonds, and each of the at least one modified nucleoside internucleotide bond is independently selected between the first and second nucleotides of the sense strand starting at the 5' end, between the second and third nucleotides of the sense strand starting at the 5' end, between the first and second nucleotides of the sense strand starting at the 3' end, between the first and second nucleotides of the antisense strand starting at the 5' end, between the second and third nucleotides of the antisense strand starting at the 5' end, between the tenth and eleventh nucleotides of the antisense strand starting at the 5' end, between the first and second nucleotides of the antisense strand starting at the 3' end, or between the second and third nucleotides of the antisense strand starting at the 3' end. Optionally, the modified nucleoside inter-bond is selected from phosphate thioside inter-bonds; Optionally, the first nucleotide of the antisense strand, starting from the 5' end, is selected from... Or a nucleotide modified with a 5'-phosphate analog; said nucleotide is selected from any nucleotide with the following structure: Wherein, Base represents a nucleoside base, which is selected from A, U, G, C or T; R represents -H, -OH, _-CH3 , _-OCH3 , -F; Optionally, the 5'-phosphate analog-modified nucleotide is selected from nucleotides modified with 5'-(E)-vinylphosphonate (5'-(E)-VP); Optionally, the first nucleotide of the antisense strand, starting from the 5' end, is selected from... Alternatively, the first nucleotide of the antisense strand, starting from the 5' end, may be selected from a nucleotide modified with 5'-(E)-VP. The double-stranded oligonucleotide according to any one of claims 6-7, characterized in that: starting from the 5' end, the antisense strand comprises at least 17 consecutive nucleotides whose nucleotides from positions 1 to 19 differ by no more than 3 nucleotides from the nucleotide sequences shown in any one of SEQ ID NO. 208-276, 307-316; and the sense strand comprises at least 17 consecutive nucleotides whose nucleotide sequences differ by no more than 3 nucleotides from the nucleotide sequences shown in any one of SEQ ID NO. 139-207, 297-306. Optionally, starting from the 5' end, the antisense strand contains no more than 3 nucleotides different from the nucleotides at positions 1-19 of any of the nucleotide sequences shown in SEQ ID NO. 208-276, 307-316; Optionally, starting from the 5' end, the antisense strand contains no more than two nucleotides differing from the nucleotide sequences shown in any of SEQ ID NO. 208-276, 307-316 in positions 1-19; Optionally, starting from the 5' end, the antisense strand contains no more than one nucleotide from positions 1 to 19 that differ from the nucleotide sequences shown in any of SEQ ID NO. 208-276, 307-316; Optionally, starting from the 5' end, the antisense strand comprises nucleotides 1-19 of the nucleotide sequence shown in any of SEQ ID NO. 208-276, 307-316; Optionally, the antisense strand is selected from any of the nucleotide sequences shown in SEQ ID NO. 208-276 and 307-316; Optionally, the double-stranded oligonucleotide is selected from the sense strand sequence shown in any one of the groups RX591001-RX591079 and the 1st to 19th nucleotide sequences of the antisense strand sequence shown in any one of the groups RX591001-RX591079, starting from the 5' end.




Optionally, the double-stranded oligonucleotide is selected from any one of the groups RX591001-RX591079. The conjugate is characterized in that it comprises the double-stranded oligonucleotide as described in any one of claims 1-9 and one or more targeting ligand groups conjugated to the double-stranded oligonucleotide; Optionally, the targeting ligand group targets the renal megalin receptor or the renal natriuretic peptide receptor; Optionally, the targeting ligand group is selected from polypeptide fragments containing at least 4 amino acids, and the targeting ligand group includes any of the polypeptide fragments shown in 1)-6) below: 1) _-X1 (KKEEE) _n - _Km- , where n＝1-5, m＝0 or 1 or 2, and _X1 is selected from any L-α amino acid or L-β amino acid other than K and E; 2)-CLPVASC-, preferably in cyclic peptide form 3)-CYFQNC-, preferably in cyclic peptide form 4)-KIDRI-; 5)-IDRI-(Ile-Asp-Arg-Ile-); 6)-dXa-Ser-dXb-X _2- dXc-Gly-Xd-Ile-Asp-Arg(Ak)-Ile-; where X ₂ is selected from any non-natural amino acid; dXa, dXb, dXc are selected from any D-type amino acid; Arg(Ak) is selected from arginine or alkylated arginine; Xd is selected from Hyp or Pro; Preferably, the targeting ligand group is selected from polypeptides containing any of the amino acid sequence segments shown in a)-g). a)-KKEEE-KKEEE-KKEEE-K-；； b)-CKKEEE-KKEEE-KKEEE-K； c)-CLPVASC-, preferably in cyclic peptide form d)-KIDRI-; e)-CYFQNCPRG-, preferably in cyclic peptide form f)-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-; g)-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-. According to claim 10, the conjugate is characterized in that the targeting ligand group is connected to the double-stranded oligonucleotide via a linker; Optionally, the structural unit of the targeting ligand group-connector in the conjugate is selected from any of the structures shown in 1)-7) below: 1)La-KKEEE-KKEEE-KKEEE-K-Laa; 2)La-CKKEEE-KKEEE-KKEEE-K-Laa; 3) La-CLPVASC-Laa, preferably a cyclic peptide. 4)La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Laa; 5) La-CYFQNCPRG-Laa; preferably cyclic peptide form. 6)La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Laa; 7) Lb-KIDRI-Laa; In formulas 1)-6), either La or Laa is a linker and the other is a terminal blocking group; the linker is a linking group containing at least one of a triazole group, a PEG unit, or an acyl group. Optionally, La is a connector, the connector structure comprising -PEG ₂ -CH ₂ CH ₂ CO- or -triazolyl-PEG _2- ; In formula 7), Lb is a linking group containing a 6-10 membered aromatic ring or heteroaromatic ring, and Laa is independently selected from an amino group or an alkyl-substituted amino group, such as -NHCH ₃ . According to claim 11, the conjugate is characterized in that the targeting ligand group is connected to the ends of the sense and/or antisense strands of the double-stranded oligonucleotide via a linker. The conjugate according to claim 11 or 12 is characterized in that: the structure of the targeting ligand group-connector in the conjugate is selected from any of the structures shown in a)-j), or a pharmaceutically acceptable salt thereof. a)-La-KKEEE-KKEEE-KKEEE-K-Lc; b)-La-CKKEEE-KKEEE-KKEEE-K-Lc; c)-La-CLPVASC-Lc; preferably cyclic peptide form d)-La-DPhe-Ser-DHyp-Cha-DAla-Gly-Hyp-Ile-Asp-Arg(Me)-Ile-Lc; e)-La-CYFQNCPRG-Lc; preferably a cyclic peptide f)-La-DPhe-Ser-DPro-Cha-DAla-Gly-Pro-Ile-Asp-Arg-Ile-Lc; g) h) i) j) Wherein, La is a linker, which is independently selected from any bond, -NH-, amide group or linking group containing at least one PEG unit; Optionally, La is a linking group comprising at least two PEG units; Optionally, La is selected from amide groups, or n is 1-3, m is 0 or 1, and Z is -CO- (carbonyl) or -NH-. Wherein, Lc is a terminal blocking group; optionally, it is a C-terminal blocking group, wherein Lc is selected from amino or alkyl-substituted amino groups, preferably _-NHCH3 . A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises a double-stranded oligonucleotide as described in any one of claims 1-9, and/or a conjugate as described in any one of claims 10-13. Use of the double-stranded oligonucleotide of any one of claims 1-9, and/or the conjugate of any one of claims 10-13, and/or the pharmaceutical composition of claim 14 in the preparation of a medicament for inhibiting URAT1 gene expression. Use of the double-stranded oligonucleotide of any one of claims 1-9, and/or the conjugate of any one of claims 10-13, and/or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating and/or preventing pathological conditions or diseases associated with URAT1 expression. The use according to claim 16 is characterized in that: the pathological condition or disease is hyperuricemia or gout. A method for inhibiting URAT1 gene expression, characterized in that it comprises administering to a subject an effective dose of the double-stranded oligonucleotide of any one of claims 1-9, and/or the conjugate of any one of claims 10-13, and/or the pharmaceutical composition of claim 14; Optionally, the subject is a human being. A method for preventing and/or treating hyperuricemia or gout, characterized in that the method comprises administering to a subject an effective dose of the double-stranded oligonucleotide of any one of claims 1-9, and/or the conjugate of any one of claims 10-13, and/or the pharmaceutical composition of claim 14.