Double-stranded oligonucleotide, conjugate, composition, and uses thereof

Abstract

Provided are a double-stranded oligonucleotide, a conjugate, a composition, and uses thereof, relating to the technical field of nucleic acid drugs. The double-stranded oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand being at least partially reverse complementary to form a duplex region having 16 to 21 base pairs; and at least one overhang is present in the double-stranded oligonucleotide, and the overhang is not located at the 5'end of the antisense strand, wherein the duplex region contains at least one nucleotide modified by [2'-R1-2'-R2].

Description

Double-stranded oligonucleotides, conjugates and compositions and their uses

[0001] Citation of relevant applications

[0002] This application claims priority and benefits to Chinese Patent Application No. 202411479748.4, filed on October 22, 2024 with the State Intellectual Property Office of the People's Republic of China, the entire contents of which are hereby incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of nucleic acid drug technology, and in particular to a double-stranded oligonucleotide, conjugates and conjugate compositions containing the same, and their uses. Background Technology

[0004] The following statements are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0005] Double-stranded oligonucleotides are a class of drugs synthesized artificially through chemical processes. They play a crucial role in molecular medicine, particularly in intervening in gene expression to treat hereditary diseases. These synthetic molecules, based on the principle of complementary base pairing, recognize and bind to target mRNA, intervening in multiple key stages of gene unwinding, replication, transcription, mRNA splicing, and even export and translation. This process effectively blocks the generation of pathogenic proteins, thereby exerting a unique mechanism for regulating the transcription and translation of disease genes at the gene level.

[0006] However, the inherent chemical properties of double-stranded oligonucleotides pose significant challenges to their clinical translation. Their complex molecular structures, large molecular weight, high polarity, and strong negative charge limit their distribution in vivo, resulting in poor pharmacokinetic characteristics and difficulty in penetrating cell membranes, particularly challenging biological barriers such as the blood-brain barrier. Furthermore, they are readily degraded by nucleases in the bloodstream and are easily recognized and excreted by the body's clearance mechanisms, such as being rapidly eliminated by the mononuclear phagocyte system in the liver and spleen, significantly reducing their bioavailability.

[0007] To overcome these obstacles, researchers are dedicated to improving the pharmacokinetic and tissue distribution properties of double-stranded oligonucleotides through sophisticated chemical modification strategies. Chemical modification can not only enhance the resistance of double-stranded oligonucleotides to nucleases and increase their affinity for target mRNA, but also significantly reduce off-target effects from non-specific binding, thereby mitigating potential toxic side effects. However, the modification process may also introduce new problems; for example, while chemical modification improves drug stability, it may simultaneously reduce the affinity between the drug and the target gene. Optimizing the modification of double-stranded oligonucleotides can improve their efficacy.

[0008] In view of this, this disclosure is hereby made. Summary of the Invention

[0009] The purpose of this disclosure is to provide double-stranded oligonucleotides, conjugates, and compositions thereof, and their uses, to improve the affinity of double-stranded oligonucleotides for target genes and their inhibitory efficiency on target genes.

[0010] To solve the above-mentioned technical problems, the present disclosure adopts the following technical solution:

[0011] In a first aspect, a double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof is provided, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 16 to 30 nucleotides, and the sense strand and the antisense strand being at least partially anticomplementary to form a double-stranded region having 16 to 21 base pairs; the double-stranded oligonucleotide having at least one dangling end, and the dangling end not being located at the 5' end of the antisense strand;

[0012] The double-stranded region contains at least one [2'-R1-2'-R2] modified nucleotide;

[0013] R1 represents a substituent group at the 2'-OH of the ribosome in the substituted nucleotide, and R2 represents a substituent group at the 2'-H of the ribosome in the substituted nucleotide; R1 and R2 are each independently selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups (i.e., a [2'-R1-2'-R2] modified nucleotide represents a nucleotide having R1 and R2 substituents at the 2' of the ribosome).

[0014] In a second aspect, a conjugate is provided, the conjugate comprising the double-stranded oligonucleotide of the first aspect or a pharmaceutically acceptable salt thereof and one or more targeted delivery ligands.

[0015] Thirdly, this disclosure provides compositions comprising the double-stranded oligonucleotides described in the first aspect or pharmaceutically acceptable salts thereof and / or conjugates described in the second aspect.

[0016] Fourthly, this disclosure provides the use of any of the following in the preparation of a medicament for the prevention and / or treatment of diseases:

[0017] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0018] (II) The conjugates described in the second aspect; and / or

[0019] (III) The composition described in the third aspect.

[0020] Fifthly, this disclosure provides the use of any of the following in the preparation of a medicament for reducing the expression or activity of a target gene:

[0021] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0022] (II) The conjugates described in the second aspect; and / or

[0023] (III) The composition described in the third aspect.

[0024] Sixthly, this disclosure provides pharmaceutical compositions comprising any one of the following and pharmaceutically acceptable excipients:

[0025] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0026] (II) The conjugates described in the second aspect; and / or

[0027] (III) The composition described in the third aspect.

[0028] In a seventh aspect, this disclosure provides a method for preventing and / or treating a disease, said method comprising contacting cells with any of the following:

[0029] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0030] (II) The conjugates described in the second aspect; and / or

[0031] (III) The composition described in the third aspect; and / or

[0032] (IV) The pharmaceutical composition described in aspect 6.

[0033] Eighthly, this disclosure provides a method for reducing the expression or activity of a target gene, said method comprising contacting a cell with any of the following:

[0034] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0035] (II) The conjugates described in the second aspect; and / or

[0036] (III) The composition described in the third aspect; and / or

[0037] (IV) The pharmaceutical composition described in aspect 6.

[0038] In a ninth aspect, this disclosure provides a kit comprising the double-stranded oligonucleotide of the first aspect or a pharmaceutically acceptable salt thereof, the conjugate of the second aspect, the composition of the third aspect and / or the pharmaceutical composition of the sixth aspect; and instructions for use optionally for the prevention and / or treatment of disease or for reducing the expression or activity of a target gene.

[0039] In a tenth aspect, this disclosure provides the double-stranded oligonucleotides of the first aspect or pharmaceutically acceptable salts thereof for the prevention and / or treatment of diseases, the conjugates of the second aspect, the compositions of the third aspect, and / or the pharmaceutical compositions of the sixth aspect.

[0040] In the eleventh aspect, this disclosure provides the double-stranded oligonucleotides of the first aspect or pharmaceutically acceptable salts thereof for reducing the expression or activity of target genes, the conjugates of the second aspect, the compositions of the third aspect, and / or the pharmaceutical compositions of the sixth aspect.

[0041] In a twelfth aspect, this disclosure provides the use of the double-stranded oligonucleotides described in the first aspect or pharmaceutically acceptable salts thereof, the conjugates described in the second aspect, the compositions described in the third aspect, and / or the pharmaceutical compositions described in the sixth aspect for the prevention and / or treatment of diseases, or for reducing the expression or activity of target genes.

[0042] Compared with the prior art, this disclosure has the following beneficial effects:

[0043] This disclosure enhances the affinity and inhibitory capacity of double-stranded oligonucleotides or their pharmaceutically acceptable salts for target genes, thereby significantly reducing the activity of target genes in subjects. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0045] Figure 1 shows the inhibitory activity of the target gene CFB in mouse primary liver cells after administration of the siRNA conjugate in Example 1.

[0046] Figure 2 shows the inhibitory activity of the target gene CFB in mouse primary liver cells after administration of the siRNA conjugate in Example 2.

[0047] Figure 3 shows the inhibitory activity of the target gene SOD1 in mouse primary liver cells after administration of the siRNA conjugate in Example 3.

[0048] Figure 4 shows the inhibitory activity of the target gene ANGPTL3 in mouse primary liver cells after administration of the siRNA conjugate in Example 4.

[0049] Figure 5 shows the inhibitory activity of the siRNA conjugate on the target gene CFB in mice in Example 5.

[0050] Figure 6 shows the inhibitory activity of the siRNA conjugate on the target gene C4B in mice in Example 6.

[0051] Figure 7 shows the inhibitory activity of the siRNA conjugate on the target gene CFB in mice in Example 7.

[0052] Figure 8 shows the inhibitory activity of the siRNA conjugate on the target gene C4B in mice in Example 8.

[0053] Figure 9 shows the inhibitory activity of the siRNA conjugate on the target gene SOD1 in mouse primary liver cells after administration in Example 9.

[0054] Figure 10 shows the inhibitory activity of the siRNA conjugate on the target gene SOD1 in mice in Example 10.

[0055] Figure 11 shows the inhibitory activity of the siRNA conjugate on the target gene ANGPTL3 in mice in Example 11.

[0056] Figure 12 shows the relative expression level of SEAP in the serum of SEAP reporter gene tool mice after administration of the siRNA conjugate in Example 12.

[0057] Figure 13 shows the relative expression level of the target gene SOD1 in mouse primary hepatocytes after administration of the siRNA conjugate described in this embodiment. Detailed Implementation

[0058] The technical solutions of this disclosure will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0059] Terminology Explanation:

[0060] Unless otherwise stated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements are consistent with the CAS edition of the periodic table and the Handbook of Chemistry and Physics, 75th edition, 1994. Furthermore, general principles of organic chemistry can be found in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0061] In this article, double-stranded oligonucleotides consist of two strands, one of which is called the antisense strand or guide strand, and the other is called the sense strand or guest strand.

[0062] In this paper, "antisense strand" includes a region that is at least partially anticomplementary to a target sequence. "Sense strand" refers to an RNA strand containing an RNA strand that is partially anticomplementary to, preferably substantially complementary to, the antisense strand.

[0063] In this paper, "partially reverse complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved, such as 3, 2, 1, or 0. "Substantially complementary" means that there are no more than two base mismatches between the two nucleotide sequences involved, such as 2, 1, or 0.

[0064] In this article, "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases of the other strand in a complementary manner.

[0065] In this paper, "target sequence" refers to the continuous nucleotide sequence that is complementary to the antisense strand of siRNA in the messenger RNA (mRNA) molecule generated during the transcription of the target gene, including portions of the sequence derived from the primary transcript (pre-mRNA) and the processed mature mRNA.

[0066] In this article, a "drooping end" (or overhang) refers to at least one non-base-paired nucleotide that protrudes from the double helix structure of a double-stranded oligonucleotide (e.g., dsRNA). For example, a drooping end is defined as the 3' end of one strand of the sense strand and / or the antisense strand extending beyond the 5' end of the other strand, or the 5' end of one strand extending beyond the 3' end of the other strand. The drooping end may contain at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. The drooping end may appear at the 5' end of the sense strand, and / or the 3' end of the sense strand, and / or the 3' end of the antisense strand.

[0067] In this document, “optionally,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0068] In this article, the term "optionally substituted" is used to define a variable that can be either unsubstituted or substituted.

[0069] In this article, the term "unsubstituted" means that the specified group does not contain substituents.

[0070] In this document, the terms “substituted,” “substituted,” and “substituted” are used interchangeably to refer to any one or more hydrogen atoms in the given structure being specifically substituented (e.g., C). 1-6 Alkyl, C 1-6 The substituted group may be replaced by an alkoxy or halogen group, provided that the normal valence of the specified atom does not exceed the valence of the substituted atom and the substitution produces a stable compound. Unless otherwise indicated, a substituted group may have one substituent at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a particular group, then the substituents may be substituted at each substituted position in the same or different manner.

[0071] In this article, the terms “each…independently selected”, “…independently selected”, and “…independently selected” are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0072] In this article, "halogen" or "halogenated" refers to any of the radioactive-stable atoms in column 7 of the periodic table, such as fluorine, chlorine, bromine, or iodine; among which, fluorine and chlorine are preferred, and fluorine is more preferred.

[0073] In this article, "hydroxyl group" refers to the -OH group.

[0074] In this text, "alkyl" refers to a fully saturated (i.e., without double or triple bonds) straight-chain or branched alkane chain. "C1-C6 alkyl" indicates an alkyl chain having 1 to 6 carbon atoms (whenever it appears in the text, numerical ranges such as "1 to 6" refer to each integer within the given range; for example, "1 to 6 carbon atoms" means that the alkyl chain can consist of 1, 2, 3, 4, 5, or 6 carbon atoms). Typical C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl. Alkyl groups can be substituted or unsubstituted. "C10-C22 alkyl" indicates an alkyl chain having 10 to 22 carbon atoms (whenever it appears in the text, the numerical range such as "10 to 22" refers to each integer within the given range; for example, "10 to 22 carbon atoms" means that an alkyl chain can consist of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbon atoms). It should be noted that this definition also covers the term "alkyl" where no numerical range is specified.

[0075] In this article, "alkoxy" refers to the formula -OR, where R is an alkyl group as defined above, such as "C1-C6 alkoxy", including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

[0076] In this article, "alkenyl" refers to an unsaturated hydrocarbon group with one or more carbon-carbon double bonds formed by removing one or more hydrogen atoms from an olefin molecule. "C10-C22 alkenyl" indicates that the alkenyl chain has 10 to 22 carbon atoms.

[0077] In this article, "alkynyl" refers to an unsaturated hydrocarbon group containing a carbon-carbon triple bond. "C10-C22 alkynyl" indicates that the alkynyl chain has 10 to 22 carbon atoms.

[0078] In this article, when a letter refers to an object or quantity, it is case-sensitive.

[0079] In this document, when referring to modifications of nucleotides, unless otherwise stated, "2'-... modification" refers to a modification present at the 2' position of the ribosome in the nucleotide. Therefore, in this document, when referring to modifications of nucleotides, unless otherwise stated, "2'-... modified nucleotide" refers to a nucleotide formed by a modification present at the 2' position of the ribosome in the nucleotide.

[0080] In this article, "2'-halogen-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosome is replaced by a halogen atom. For example, a 2'-F-modified nucleotide.

[0081] In this article, "2'-deoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a hydrogen atom.

[0082] RNAi agents are substances that induce RNA interference, and their core component is typically short double-stranded RNA. Common RNAi agents include small interfering RNA (siRNA), shRNA, and miRNA. After entering the cell, the RNAi agent is first cleaved by the Dicer enzyme, and the resulting small interfering RNA (siRNA) then integrates into the RISC complex. RISC uses siRNA as a guide to find and bind to complementary mRNA sequences. Once a match is found, the target mRNA is cleaved and degraded, preventing its translation into protein, thereby achieving gene silencing. In this document, the term RNAi agent may refer to the double-stranded oligonucleotide or its conjugate described herein.

[0083] In this article, "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, containing 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 the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and its conversion into proteins.

[0084] In this document, the terms "conjugation," "linkage," and "conjugation-linkage" are used interchangeably to refer to the covalent connection between two or more chemical parts, each with a specific function; correspondingly, a "conjugated compound" refers to a compound formed by the covalent connection of two or more chemical parts. Further, a "pharmaceutical conjugation" refers to a compound formed by the covalent attachment of one or more chemical parts with specific functions to an active pharmaceutical ingredient. In this document, sometimes, particularly in embodiments, the "pharmaceutical conjugation" of this disclosure is also referred to as a "conjugation," "double-stranded oligonucleotide conjugation," or "siRNA conjugation." The term "pharmaceutical conjugation" should be understood, depending on the context, as a general term for pharmaceutical conjugations or a specific pharmaceutical conjugation represented by a particular structural formula.

[0085] In this document, the terms "targeted delivery ligand," "delivery ligand," "ligand," "targeted delivery vector," "delivery vector," and "vector" are used interchangeably and generally refer to any compound or molecule capable of covalently or otherwise chemically binding to a bioactive substance (such as an oligonucleotide). In some embodiments, the ligand is capable of interacting directly or indirectly with another compound, such as a receptor. The receptor interacting with the ligand may be present on the cell surface or, alternatively, may be an intracellular and / or intercellular receptor. The interaction between the ligand and the receptor may result in a biochemical reaction or may simply be a physical interaction or binding.

[0086] In this document, "subject" generally refers to a human or non-human animal (including mammals) for the diagnosis, prognosis, improvement, prevention, and / or treatment of a disease, and is not intended to be limited to any particular type of subject in this disclosure; or, in this document, the term "subject" refers to any animal being examined, studied, or treated, and is not intended to be limited to any particular type of subject in this disclosure. Examples of subjects include, but are not limited to, humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (horses, cattle, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents, and adult subjects. Subjects include animal disease models. In some embodiments of this disclosure, humans are preferred subjects, while 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 this document, the term "subject" is used interchangeably with "individual."

[0087] In this disclosure, the terms “comprising,” “including,” “having,” “may,” “containing,” and variations thereof are generally intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The term “consisting of” generally indicates that no other components can exist (or similarly, features, integers, steps, etc.). Unless the context clearly specifies otherwise, nouns without a defined quantity also include plural indicators.

[0088] In this document, "pharmaceutical composition" can refer to a composition containing an active ingredient for the treatment of a disease or for use in in vitro cell culture experiments. In some embodiments of this disclosure, 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, the composition is prepared by uniformly and sufficiently combining the active pharmaceutical ingredient with a liquid excipient, a finely chopped solid excipient, or both.

[0089] In this article, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or with subjects (e.g., mammals) treated with it.

[0090] In this document, "pharmaceutically acceptable salt" refers to a compound obtained by modifying the compound of this application through salt formation with an acid or a base. Examples of pharmaceutically acceptable salts include, but are not limited to, sodium salts, potassium salts, magnesium salts, calcium salts, triethylamine salts, spermine salts, and ammonium salts.

[0091] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. In addition to any conventional excipients, the scope of incompatibility with the double-stranded oligonucleotides of this disclosure or conjugates containing them, such as any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition, is also within the scope of this disclosure.

[0092] In this document, the term "treatment" means used to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to the disease but not yet diagnosed with it; (b) inhibition of a disease, such as blocking disease progression; or (c) relief of a disease, such as reducing or improving symptoms associated with the disease. As used herein, "treatment" encompasses any administration of an active agent (e.g., siRNA or its conjugates), pharmaceutical composition, or drug of this disclosure to an individual to treat, cure, relieve, improve, reduce, or inhibit a disease in that individual, including but not limited to administration of a drug containing the siRNA or siRNA conjugates described herein to an individual in need. The terms "treatment," "relief," or "improvement" are used interchangeably.

[0093] In this article, the terms “prevention” and “protection” are used interchangeably to refer to obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” the double-stranded oligonucleotide described herein or its pharmaceutically acceptable salts, conjugates, or compositions may be given to subjects at risk of developing a specific disease, or to subjects who report one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0094] In this document, the terms “reduction,” “inhibition,” “decrease,” “silence,” “downregulation,” “blockade,” and other similar terms are used interchangeably and include any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. This control level can be any type of control level used in the art, such as baseline levels before administration or levels determined from untreated or controlled (e.g., buffer-only control or inert agent control) subjects, cells, or samples.

[0095] In this document, the term "contact" generally refers to two or more substances of different types coming into contact with each other in any order, in any manner, and for any duration. Contact can occur in vivo, ex vivo, or in vitro. In some embodiments, it can refer to direct contact of the RNAi agent or composition of this disclosure with cells or tissues. In other embodiments, the term refers to indirect contact of the RNAi agent or composition of this disclosure with cells or tissues.

[0096] In this document, "effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For preventative use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of symptoms, including the symptoms, their complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the symptoms. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various symptoms associated with the target genes, target mRNAs, or target proteins of this disclosure, or improving one or more symptoms of said symptoms, reducing the dosage of other agents required to treat the symptoms, enhancing the efficacy of another agent, and / or delaying the progression of the symptoms associated with the target genes, target mRNAs, or target proteins of this disclosure in patients.

[0097] In this document, "double-stranded oligonucleotides" and "conjugates" can be obtained using conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the double-stranded oligonucleotides described herein using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and for introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.

[0098] In this article, the term "chemical modification" or "modification" includes all alterations to nucleic acids by chemical means, such as the addition or removal of chemical parts, or the substitution of one chemical part for another.

[0099] In this document, "base" includes any known DNA and RNA bases, base analogues such as purines or pyrimidines, and also includes natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues. As an example, "base" includes natural bases or synthetic nucleoside base analogues, including but not limited to adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), inosine (I), 5-nitroindole (5-NI), 3-nitropyrrole (3-NP), 2-aminopurine (2-AP), and 2,6-diaminopurine (2,6-DAP). In this article, the terms “base” and “nucleoside base” are used interchangeably.

[0100] In this article, the structural formulas of "compounds," "ligands," and "supports" contain bonds. This indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. Although all the above structural formulas are shown in certain isomer forms for simplicity, this disclosure can include all isomers, such as stereoisomers, tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0101] In this article, the term "stereoisomer" refers to compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers, etc.

[0102] In this article, the term "chirality" refers to a molecule that has the property that it cannot be superimposed on its mirror image; while "chirality" refers to a molecule that can be superimposed on its mirror image.

[0103] In this article, the term "enantiomer" refers to two non-overlapping but mirror-image isomers of a compound.

[0104] In this article, the term "diastereomer" refers to a stereoisomer with two or more chiral centers whose molecules are not mirror images of each other. Diastereomers possess different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0105] In this article, the abbreviations for nucleotides are explained as follows: BNA stands for bridged nucleic acid, GNA stands for glycol nucleic acid, HNA stands for hexitol nucleic acid, CeNA stands for cyclohexenyl nucleic acid, LNA stands for locked nucleic acid, and UNA stands for unlocked nucleic acid.

[0106] Unless otherwise stated, in this document, “Base” represents a nucleoside base or base, such as A, U, G, C or T; and “Z” represents a hydroxyl (-OH) or thiol (-SH).

[0107] Unless otherwise specified, singular terms encompass plural terms, and plural terms encompass singular terms. Unless otherwise specified, the words "a" or "an" mean "at least one" or "at least one". Unless otherwise specified, the use of "or" means "and / or".

[0108] For purposes of description and disclosure, all patents, patent applications, and other identified publications are expressly incorporated herein by reference. These publications are provided solely because their publication predates the filing date of this application. All statements regarding the dates of these documents or representations of their contents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. Furthermore, in any country, any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0109] Double-stranded oligonucleotides:

[0110] In a first aspect, a double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof is provided, said double-stranded oligonucleotide comprising a sense strand and an antisense strand.

[0111] The positive chain has 16 to 30 nucleotides, such as, but not limited to, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0112] The antisense strand has 16 to 30 nucleotides, such as, but not limited to, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0113] The sense and antisense strands of the double-stranded oligonucleotide are at least partially anticomplementary to form a double-stranded region having 16 to 21 base pairs, for example, but not limited to, forming a double-stranded region having 16, 17, 18, 19, 20, or 21 base pairs; the double-stranded oligonucleotide has at least one dangling end, for example, one or two dangling ends, and the dangling end is not located at the 5' end of the antisense strand.

[0114] The double-stranded oligonucleotide contains at least one [2'-R1-2'-R2] modified nucleotide in its double-stranded region. The [2'-R1-2'-R2] modified nucleotide comprises... Base is selected from nucleoside bases (e.g., A, U, G, C, or T), wherein R1 represents a substituent group of the 2'-OH ribose ring in the substituted nucleotide, and R1 is selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups; R2 represents a substituent group of the 2'-H ribose ring in the substituted nucleotide, and R2 is selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups. In a specific embodiment, the structural formula of the [2'-R1-2'-R2] modified nucleotide is as follows: Base is selected from nucleoside bases (e.g., A, U, G, C, or T), Z represents -OH or -SH, where R1 represents a substituent group of the 2'-OH ribose ring in the nucleotide, R1 being selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups; R2 represents a substituent group of the 2'-H ribose ring in the nucleotide, R2 being selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups. For example, the structural formula of a [2'-R1-2'-R2] modified nucleotide is selected from... Base is selected from nucleoside bases (e.g., A, U, G, C, or T), where R1 represents a substituent group of the 2'-OH ribose ring in the substituted nucleotide, and R1 is selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups; R2 represents a substituent group of the 2'-H ribose ring in the substituted nucleotide, and R2 is selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups.

[0115] In some alternative embodiments, in the double-stranded region, at least one nucleotide from the antisense strand at positions 2, 6, 9, 12, 14, and 16 (starting from the 5' end) and the sense strand that are base-complementarily paired with nucleotides at positions 10-13 and 15 (starting from the 5' end) of the antisense strand is selected from nucleotides modified with [2'-R1-2'-R2].

[0116] In some specific embodiments, the second nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0117] In some specific embodiments, the 6th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0118] In some specific embodiments, the 9th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0119] In some specific embodiments, the 12th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0120] In some specific embodiments, the 14th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0121] In some specific embodiments, the 16th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0122] In some specific embodiments, the nucleotides at positions 14 and 16 of the antisense strand, starting from the 5' end, are selected from nucleotides modified with [2'-R1-2'-R2].

[0123] In some specific embodiments, the nucleotide in the sense strand that is base-complementary to the 10th nucleotide starting from the 5' end in the antisense strand is selected from nucleotides modified with [2'-R1-2'-R2].

[0124] In some specific embodiments, the nucleotide in the sense strand that is base-complementarily paired with the 11th nucleotide starting from the 5' end in the antisense strand is selected from nucleotides modified with [2'-R1-2'-R2].

[0125] In some specific embodiments, the nucleotide in the sense strand that pairs with the 12th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2].

[0126] In some specific embodiments, the nucleotide in the sense strand that is base-complementary to the 13th nucleotide starting from the 5' end in the antisense strand is selected from nucleotides modified with [2'-R1-2'-R2].

[0127] In some specific embodiments, the nucleotide in the sense strand that is base-complementary to the 15th nucleotide starting from the 5' end in the antisense strand is selected from nucleotides modified with [2'-R1-2'-R2].

[0128] In some alternative embodiments, at least five nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, are each independently selected from nucleotides modified with [2'-R1-2'-R2] or nucleotides modified with 2'-fluorine, and the antisense strand contains no more than five nucleotides modified with 2'-fluorine; and / or, at least four nucleotides in the sense strand that are base-paired with nucleotides at positions 10-13 and 15 of the antisense strand, starting from the 5' end, are each independently selected from nucleotides modified with [2'-R1-2'-R2] or nucleotides modified with 2'-fluorine, and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0129] Furthermore, among the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand (starting from the 5' end) and the nucleotides at positions 10-13 and 15 of the sense strand (starting from the 5' end) that are base-complementary, at least one nucleotide is selected from a nucleotide modified with [2'-R1-2'-R2].

[0130] In some alternative embodiments, the second nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 6, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0131] In some alternative embodiments, the 6th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]; at least four of the nucleotides at positions 2, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0132] In some alternative embodiments, the 9th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]; at least four of the nucleotides at positions 2, 6, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0133] In some alternative embodiments, the 12th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]; at least four of the nucleotides at positions 2, 6, 9, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0134] In some alternative embodiments, the 14th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 9, 12, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0135] In some alternative embodiments, the 16th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 9, 12, and 14 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0136] In some alternative embodiments, the 14th and 16th nucleotides of the antisense strand, starting from the 5' end, are selected from nucleotides modified with [2'-R1-2'-R2], and the nucleotides at positions 2, 6, 9, and 12 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0137] In some alternative embodiments, the 9th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0138] In some alternative embodiments, the nucleotide in the sense strand that is complementary to the 10th nucleotide starting from the 5' end in the antisense strand is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides that are complementary to the 11th-13th and 15th nucleotides starting from the 5' end in the antisense strand are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0139] In some alternative embodiments, the nucleotide in the sense strand that pairs with the 11th nucleotide starting from the 5' end of the antisense strand via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the antisense strand that pair with the 10th, 12th-13th and 15th nucleotides starting from the 5' end via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0140] In some alternative embodiments, the nucleotide in the sense strand that pairs with the 12th nucleotide starting from the 5' end of the antisense strand through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the antisense strand that pair with the 10th-11th, 13th and 15th nucleotides starting from the 5' end through base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0141] In some alternative embodiments, the nucleotide in the sense strand that pairs with the 13th nucleotide starting from the 5' end of the antisense strand via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the antisense strand that pair with the 10th-12th and 15th nucleotides starting from the 5' end via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0142] In some alternative embodiments, the nucleotide in the sense strand that pairs with the 15th nucleotide starting from the 5' end of the antisense strand through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the antisense strand that pair with the 10th-13th nucleotides starting from the 5' end through base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0143] In some alternative embodiments, in the double-stranded oligonucleotide, the R1 in each [2'-R1-2'-R2] modified nucleotide is independently selected from halogens or optionally substituted C1-C6 alkoxy groups.

[0144] In some alternative embodiments, in the double-stranded oligonucleotide, the R2 in each [2'-R1-2'-R2] modified nucleotide is independently selected from halogens or optionally substituted C1-C6 alkoxy groups.

[0145] In some alternative embodiments, in each of the [2'-R1-2'-R2] double-stranded oligonucleotides, R1 is independently selected from F or methoxy, and R2 is independently selected from F or methyl.

[0146] In some alternative embodiments, each [2'-R1-2'-R2]-modified nucleotide in the double-stranded oligonucleotide is independently selected from [2'-F-2'-F]-modified nucleotides, [2'-F-2'-CH3]-modified nucleotides (also known as [2'-F-2'-Me]-modified nucleotides), or [2'-OCH3-2'-CH3]-modified nucleotides (also known as [2'-OMe-2'-Me]-modified nucleotides). The [2'-F-2'-F]-modified nucleotides have… Wherein, Base is selected from nucleoside bases (e.g., A, U, G, C, or T). Wherein, the [2'-F-2'-CH3] modified nucleotide has... Wherein, Base is selected from nucleoside bases (e.g., A, U, G, C, or T). Wherein, the nucleotide modified with [2'-OCH3-2'-CH3] has... Wherein, Base is selected from nucleoside bases (e.g., A, U, G, C, or T). In some embodiments, the structural formula of the [2'-F-2'-F]-modified nucleotide is as follows: Wherein, Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z represents -OH or -SH. In some embodiments, the structural formula of the [2'-F-2'-CH3]-modified nucleotide is as follows: Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z represents -OH or -SH. In some embodiments, the structural formula of the [2'-OCH3-2'-CH3] modified nucleotide is as follows: Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z represents -OH or -SH. In some embodiments, the structural formula of the [2'-F-2'-F]-modified nucleotide is... Wherein, Base is selected from nucleoside bases (e.g., A, U, G, C, or T). In some embodiments, the structural formula of the [2'-F-2'-CH3] modified nucleotide is selected from... Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T). In some embodiments, the structural formula of the [2'-OCH3-2'-CH3] modified nucleotide is as follows: Base is selected from nucleoside bases (e.g., A, U, G, C, or T).

[0147] In some alternative embodiments, the double-stranded oligonucleotide has at least one overhang (e.g., one overhang, two overhangs, etc.), each overhang independently consisting of 1-3 modified nucleotides, and at least one [2'-R1-2'-R2] modified nucleotide is present in the overhang. In some alternative embodiments, the [2'-R1-2'-R2] modified nucleotide may not be present in the overhang.

[0148] In some alternative embodiments, the double-stranded oligonucleotide has two dangling ends, which are located at the 3' end of the antisense strand and the 3' end of the sense strand, respectively.

[0149] In some specific embodiments, the double-stranded oligonucleotide has a dangling end, and the dangling end is located at the 3' end of the antisense strand.

[0150] In some alternative embodiments, the double-stranded oligonucleotide has a dangling end located at the 3' end of the positive strand.

[0151] In some specific embodiments, the double-stranded oligonucleotide has a dangling end, and the dangling end is located at the 5' end of the positive strand.

[0152] In some alternative embodiments, the overhang contains 1-3 [2'-R1-2'-R2] modified nucleotides or is composed of 1-3 [2'-R1-2'-R2] modified nucleotides.

[0153] In some alternative embodiments, the overhang comprises or consists of two [2'-R1-2'-R2] modified nucleotides.

[0154] In some specific embodiments, the overhanging end comprises or consists of two [2'-F-2'-CH3] modified nucleotides.

[0155] In some embodiments, the double-stranded oligonucleotide contains at least three different modified nucleotides. In some alternative embodiments, the double-stranded oligonucleotide contains at least four different modified nucleotides.

[0156] In some optional embodiments, the double-stranded oligonucleotide contains at least three different modified nucleotides, wherein the double-stranded oligonucleotide includes a first modified nucleotide, a second modified nucleotide, and a third modified nucleotide. In some optional embodiments, the double-stranded oligonucleotide contains at least four different modified nucleotides; wherein the double-stranded oligonucleotide includes a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, and a fourth modified nucleotide.

[0157] In some embodiments, the double-stranded oligonucleotide contains at least three different modified nucleotides, each of which may be independently selected from a first modified nucleotide, a second modified nucleotide, and a third modified nucleotide. In some alternative embodiments, the double-stranded oligonucleotide contains at least four different modified nucleotides, each of which may be independently selected from a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, and a fourth modified nucleotide.

[0158] The first modified nucleotide is selected from nucleotides modified with [2'-R1-2'-R2]. In some alternative embodiments, the first modified nucleotide includes at least one of nucleotides modified with [2'-F-2'-F], [2'-F-2'-CH3], or [2'-OCH3-2'-CH3].

[0159] The second modified nucleotide is selected from 2'-O-(CH2). n -R3 modified nucleotides have the following structural formula: Wherein, n is selected from an integer from 0 to 3 (e.g., 0, 1, 2, or 3), Base is selected from a nucleoside base (e.g., A, U, G, C, or T), Z represents -OH or -SH, and R3 is selected from optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, or... R 3a R 3b and R 3c Each is independently selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; for example In some embodiments, R3 is selected from C1-C6 alkyl groups optionally substituted with one or more halogens (e.g., F); C1-C6 alkoxy groups optionally substituted with one or more halogens (e.g., F); or Where R 3a R 3b and R 3c Each is independently selected from C1-C6 alkyl or C1-C6 alkoxy groups that are optionally substituted with one or more halogens (e.g., F).

[0160] In some optional embodiments, the second modified nucleotide includes at least one selected from the following: a nucleotide modified with 2'-O-CH3, a nucleotide modified with 2'-O-CH2-O-CH2-CH3, a nucleotide modified with 2'-O-CH2-O-CH2-CF3, a nucleotide modified with 2'-O-CH2-CH2-O-CH3 (also known as 2'-O-methoxyethyl, abbreviated as 2'-O-MOE), a nucleotide modified with 2'-O-TBDMS, a nucleotide modified with 2'-O-TIPS, or a nucleotide modified with 2'-O-TOM. The structural formula of TBDMS is [insert structural formula here]. The structural formula of TIPS is: The structural formula of TOM is: In some embodiments, the structural formula of the nucleotide modified with 2'-O-methyl (2'-O-CH3) is as follows: Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z represents -OH or -SH; for example In some embodiments, the structural formula of the nucleotide modified with 2'-O-MOE is as follows: Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z represents -OH or -SH; for example

[0161] The third modified nucleotide includes at least one of a 2'-fluoro (abbreviated 2'-F) modified nucleotide or a 2'-deoxy modified nucleotide. In some embodiments, the 2'-F modified nucleotide has Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T). In some embodiments, the structural formula of the 2'-F modified nucleotide is: Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z represents -OH or -SH; for example In some embodiments, the 2'-deoxy-modified nucleotide has Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T). In some embodiments, the structural formula of the 2'-deoxy-modified nucleotide is [structure omitted]. Where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z represents -OH or -SH; for example

[0162] The fourth modified nucleotide includes (abbreviated as RNA) (abbreviated as BNA) (abbreviated as CeNA) (abbreviated as HNA) At least one of (2'-O-allyl, 2'-O-NMA, 2'-O-AP or 2'-O-C10-C22 alkyl-modified nucleotides) or acyclic modified nucleotides, wherein each Base is independently selected from nucleoside bases A, U, G, C or T, and each Z is independently selected from -OH or -SH.

[0163] In some embodiments, the fourth modified nucleotide includes (Phosphothiophosphate-modified ribonucleotides), wherein each Base is independently selected from nucleoside bases (e.g., A, U, G, C, or T), and Z is -SH; (abbreviated as BNA), where Base is selected from nucleoside bases (e.g., A, U, G, C or T), and Z is selected from -OH or -SH; (abbreviated as CeNA), where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z is selected from -OH or -SH; (abbreviated as HNA), where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z is selected from -OH or -SH; (nucleotides modified with 2'-O-allyl, 2'-O-NMA, 2'-O-AP or 2'-O-C10-C22 alkyl), wherein Base is selected from nucleoside bases (e.g., A, U, G, C or T) and Z is selected from -OH or -SH; or at least one of acyclic modified nucleotides, wherein Base is selected from nucleoside bases (e.g., A, U, G, C or T) and Z is selected from -OH or -SH.

[0164] In some embodiments, the fourth modified nucleotide includes the following: (Phosphothiophosphate-modified ribonucleotides), where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z is -SH; (abbreviated as BNA), where R 4a and R 4b Each is independently selected from H or optionally substituted C1-C3 alkyl groups, j1 is selected from 1-3, Base is selected from nucleoside bases (e.g. A, U, G, C or T), and Z is selected from -OH or -SH; (abbreviated as CeNA), where Base is selected from nucleoside bases (e.g., A, U, G, C or T), and Z is selected from -OH or -SH; (abbreviated as HNA), where Base is selected from nucleoside bases (e.g., A, U, G, C or T), and Z is selected from -OH or -SH; R8 is selected from Optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl, or optionally substituted C10-C22 alkynyl, where Base is selected from nucleoside bases (e.g., A, U, G, C, or T) and Z is selected from -OH or -SH, such as nucleotides modified with 2'-O-allyl, 2'-O-NMA, 2'-O-AP, or 2'-O-C10-C22 alkyl; or acyclic nucleotides.

[0165] In some alternative implementations, R 4a and R 4b Each is independently selected from H or optionally substituted C1-C3 alkyl groups; j1 is selected from 1, 2, or 3. In some alternative embodiments, R 4a and R 4b All are selected from H, and j1 is selected from 1. In some alternative implementations, BNA is selected from (abbreviated as LNA), where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z is selected from -OH or -SH. In some alternative embodiments, R 4a and R 4b Both are selected from H, and j1 is selected from 1. In some alternative implementations, BNA is selected from (abbreviated as cEt). ), where Base is selected from nucleoside bases (e.g., A, U, G, C or T), and Z is selected from -OH or -SH.

[0166] In some alternative implementations, R8 is selected from... Optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl, or optionally substituted C10-C22 alkynyl.

[0167] The acyclic modified nucleotides include At least one of the following, wherein Base is selected from nucleoside bases (e.g., A, U, G, C or T), and Z is selected from -OH or -SH.

[0168] In some alternative embodiments, the acyclic modified nucleotide comprises: Among them, R 5a and R 5b Each is independently selected from H or optionally substituted C1-C3 alkyl groups, j2 is selected from 1-3, j3 is selected from 1-4, j4 is selected from 1-4, Base is selected from nucleoside bases (e.g. A, U, G, C or T), and Z is selected from -OH or -SH; Wherein, R6 is selected from H or an optionally substituted C1-C3 alkyl group, Base is selected from a nucleoside base (e.g., A, U, G, C, or T), and Z is selected from -OH or -SH; or, R7 is selected from H, optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl or optionally substituted C10-C22 alkynyl, Base is selected from nucleoside bases (e.g. A, U, G, C or T), and Z is selected from -OH or -SH.

[0169] In some alternative implementations, R 5a and R 5b Each is independently selected from H or optionally substituted C1-C3 alkyl groups, j2 is selected from 1, 2 or 3; j3 is selected from 1, 2, 3 or 4; j4 is selected from 1, 2, 3 or 4.

[0170] In some alternative implementations, j2 is selected from 1.

[0171] In some alternative implementations, j3 is selected from 2. In some alternative implementations, j3 is selected from 2, and at least one R 5a Selected from optionally substituted C1-C3 alkyl groups. In some alternative embodiments, j3 is selected from 2, and one R 5a Selected from H, another R 5a Selected from optionally substituted C1-C3 alkyl groups. In some alternative embodiments, R 5a Selected from H or methyl.

[0172] In some alternative implementations, j4 is selected from 2. In some alternative implementations, j4 is selected from 2, and R 5b All are selected from H.

[0173] In some alternative implementations, include At least one of the following, wherein each Base is independently selected from nucleoside bases A, U, G, C or T, and each Z is independently selected from -OH or -SH.

[0174] In some alternative embodiments, R6 is selected from H or optionally substituted C1-C3 alkyl groups. In some alternative embodiments, R6 is selected from H.

[0175] In some alternative implementations, include (abbreviated as GNA), where Base is selected from nucleoside bases (e.g., A, U, G, C, or T), and Z is selected from -OH or -SH. In some alternative embodiments, GNA includes (abbreviated as (S)-GNA) or At least one of (abbreviated as (R)-GNA), wherein each Base is independently selected from nucleoside bases A, U, G, C or T, and each Z is independently selected from -OH or -SH.

[0176] In some alternative embodiments, R7 is selected from H, optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl, or optionally substituted C10-C22 alkynyl. In some alternative embodiments, R7 is selected from H, optionally substituted C10-C22 alkyl, C10-C22 alkenyl, or C10-C22 alkynyl. In some alternative embodiments, R7 is selected from H, optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl, or optionally substituted C10-C22 alkynyl.

[0177] In some alternative implementations, include (abbreviated as UNA) At least one of the following, wherein each Base is independently selected from nucleoside bases A, U, G, C or T, and each Z is independently selected from -OH or -SH.

[0178] In some alternative embodiments, the fourth modified nucleotide includes At least one of the following, wherein each Base is independently selected from nucleoside bases A, U, G, C, or T, and each Z is independently selected from -OH or -SH. In some alternative embodiments, the fourth modified nucleotide includes at least one of LNA, CeNA, GNA, or UNA.

[0179] In some alternative embodiments, the fourth modified nucleotide includes (Phosphothiophosphate-modified ribonucleotides, where Z is -SH); At least one of the following, wherein each Base is independently selected from nucleoside bases A, U, G, C, or T, and each Z is independently selected from -OH or -SH. In some alternative embodiments, the fourth modified nucleotide includes at least one of LNA, CeNA, GNA, or UNA.

[0180] In some alternative embodiments, the double-stranded oligonucleotide further includes a 5'-terminal phosphorylation modification, which includes at least one of 5'-(E)-vinylphosphonate (5'-(E)-VP) modification, 5'-methylphosphonate (5'-MP) modification, (S)-5'-C-methyl analog modification, and 5'-thiophosphate (5'-PS) modification or analogues thereof.

[0181] In some alternative embodiments, the antisense strand comprises at least three modified nucleotides, each of which may be independently selected from a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, or a fourth modified nucleotide.

[0182] In some alternative embodiments, each nucleotide of the antisense strand is independently selected from [2'-F-2'-F] modified nucleotides, [2'-F-2'-Me] modified nucleotides, [2'-OMe-2'-Me] modified nucleotides, 2'-O-CH3 modified nucleotides, 2'-O-MOE modified nucleotides, 2'-F modified nucleotides, 2'-deoxy modified nucleotides, LNA, CeNA, or RNA (including phosphate thioester modified ribonucleotides).

[0183] In some alternative embodiments, when the modification of the nucleotide on the antisense strand includes at least one of [2'-F-2'-F] modification, 2'-deoxy modification, or RNA (including ribonucleotides modified with thiophosphate), it is located at at least one of the 2nd, 4th, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand starting from the 5' end.

[0184] In some alternative embodiments, when the modification of the nucleotide on the antisense strand includes at least one of [2'-F-2'-Me] modification or [2'-OMe-2'-Me] modification, it is located at at least one of the 1st to 3rd positions of the antisense strand starting from the 3' end.

[0185] In some alternative embodiments, when the modification of the nucleotide on the antisense strand includes at least one of 2'-O-MOE modification, LNA, or CeNA, it is located at one or more positions 5-10, 15, 17-19 of the antisense strand starting from the 5' end.

[0186] In some alternative embodiments, the modification of the nucleotide on the antisense strand is selected from at least 10 positions of the antisense strand, starting from the 5' end, when the 2'-O-methyl modification is performed. These positions are the 1st, 3rd-5th, 7th-13th, 15th, and 17th-19th positions.

[0187] In some alternative embodiments, the modification of the nucleotides on the antisense strand is selected from 2'-F modifications, and is located at at least four of the 2nd, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand starting from the 5' end.

[0188] In some alternative implementations, the antisense chain contains one or more of the following features 1)-6):

[0189] Feature 1) The first nucleotide of the antisense strand, starting from the 5' end, has at least one modification selected from 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methyl analog, and 5'-thiophosphate (5'-PS) modification or analogs thereof. Further, the first nucleotide of the antisense strand, starting from the 5' end, has a 5'-(E)-vinylphosphonate modification. In some embodiments, the first nucleotide of the antisense strand, starting from the 5' end, also has a 2'-O-methyl modification.

[0190] Feature 2) The nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, include at least one of a [2'-F-2'-F] modified nucleotide, a 2'-deoxy modified nucleotide, or RNA (including phosphate thioester modified ribonucleotides), and the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, contain at least three 2'-F modified nucleotides (e.g., three, four, or five). The antisense strand contains at least one of the following nucleotides at positions 2, 6, 9, 12, 14, and 16 (starting from the 5' end): a nucleotide modified with [2'-F-2'-F] or a nucleotide modified with 2'-deoxy. Furthermore, the antisense strand contains at least three nucleotides modified with 2'-F at positions 2, 6, 9, 12, 14, and 16 (starting from the 5' end), and the remaining nucleotides at positions 2, 6, 9, 12, 14, and 16 are nucleotides modified with 2'-O-methyl. Further, the nucleotide at position 14 (starting from the 5' end) of the antisense strand is a nucleotide modified with [2'-F-2'-F].

[0191] Feature 3) The nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include a first modifying nucleotide, a second modifying nucleotide, or a fourth modifying nucleotide (e.g., At least one of the following: (or acyclic modified nucleotides). The nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include the first modified nucleotide, the second modified nucleotide, and... (wherein each group is as defined above) or at least one of the acyclic modified nucleotides. Further, the nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of the following: 2'-O-MOE modified nucleotide, 2'-O-TBDMS modified nucleotide, [2'-F-2'-Me] modified nucleotide, [2'-OMe-2'-Me] modified nucleotide, GNA, UNA, LNA, or cEt, and the other nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from 2'-O-methyl modified nucleotides. In some embodiments, the nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include a 2'-O-MOE-modified nucleotide, and the other nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from 2'-O-methyl-modified nucleotides.

[0192] Feature 4) At least one nucleotide at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, is selected from GNA or UNA. In some embodiments, at least one nucleotide at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, is selected from GNA or UNA, and the other nucleotides at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3 modified nucleotides. In some embodiments, at least one nucleotide at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, is selected from GNA or UNA, and the other nucleotides at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3 modified nucleotides or 2'-F modified nucleotides.

[0193] Feature 5) The nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of a 2'-O-MOE-modified nucleotide, a 2'-O-TBDMS-modified nucleotide, a [2'-OMe-2'-Me]-modified nucleotide, or an LNA, and the other nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3-modified nucleotides. In some embodiments, the nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of a 2'-O-MOE-modified nucleotide, a 2'-O-TBDMS-modified nucleotide, a [2'-OMe-2'-Me]-modified nucleotide, or an LNA, and the other nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3-modified nucleotides or 2'-F-modified nucleotides.

[0194] Feature 6) The nucleotides at positions 11-13, starting from the 5' end of the antisense strand, are each independently selected from at least one of a 2'-O-CH3 modified nucleotide or a 2'-F modified nucleotide. Further, the nucleotides at positions 11-13, starting from the 5' end of the antisense strand, are all 2'-O-CH3 modified nucleotides, or the nucleotides at positions 11 and 13, starting from the 5' end of the antisense strand, are selected from 2'-O-CH3 modified nucleotides, and the nucleotide at position 12 is selected from a 2'-F modified nucleotide.

[0195] In some embodiments, starting from the 5' end, the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from nucleotides modified with [2'-F-2'-F], nucleotides modified with 2'-F, or nucleotides modified with 2'-O-CH3, and contain at least three nucleotides modified with 2'-F (e.g., three, four, or five). Preferably, the nucleotides at positions 2, 6, and 12 are nucleotides modified with 2'-F, and the nucleotides at positions 9, 14, or 16 are selected from nucleotides modified with [2'-F-2'-F], nucleotides modified with 2'-F, or nucleotides modified with 2'-O-CH3.

[0196] The nucleotides at positions 1, 3-5, 7-8, 10-11, 13, and 15 of the antisense strand are selected from nucleotides modified with 2'-O-MOE or nucleotides modified with 2'-O-CH3. Preferably, the nucleotide at position 15 is a nucleotide modified with 2'-O-MOE, and the nucleotides at positions 1, 3-5, 7-8, 10-11, and 13 are all nucleotides modified with 2'-O-CH3.

[0197] The nucleotides at positions 17-19 or 17-21 are selected from nucleotides modified with 2'-O-MOE, [2'-OMe-2'-Me], [2'-F-2'-Me], or nucleotides modified with 2'-O-CH3; preferably, the nucleotide at position 17 is a nucleotide modified with 2'-O-CH3, and the nucleotides at positions 18 and 19 are selected from nucleotides modified with [2'-F-2'-Me], [2'-OMe-2'-Me], or nucleotides modified with 2'-O-CH3; or preferably, the nucleotides at positions 17-19 are nucleotides modified with 2'-O-CH3, and the nucleotides at positions 20 and 21 are selected from nucleotides modified with [2'-F-2'-Me], [2'-OMe-2'-Me], or nucleotides modified with 2'-O-CH3.

[0198] Optionally, starting from the 5' end, the antisense strand further comprises one or more of the following: at least one 2'-deoxy modified nucleotide at position 9, 14, or 16; at least one of GNA or UNA in positions 5-7; 5'-(E)-vinylphosphonate modified nucleotide at position 1; and at least one of 2'-O-TBDMS modified nucleotide, GNA, UNA, LNA, or cEt in positions 3-5, 7-11, 13-15, 17-19, or 17-21.

[0199] In some embodiments, starting from the 5' end, the nucleotides at positions 2, 6, and 12 of the antisense strand are 2'-F modified nucleotides, at least one nucleotide at position 9 and 14 is selected from [2'-F-2'-F] modified nucleotides, and the nucleotide at position 16 is selected from either a 2'-F modified nucleotide or a 2'-O-CH3 modified nucleotide.

[0200] In some alternative embodiments, the antisense strand has nucleotides 2, 6, and 12 starting from the 5' end that are 2'-F modified nucleotides, at least one of the nucleotides at positions 14 and 16 is a [2'-F-2'-F] modified nucleotide, a 2'-F modified nucleotide, or a 2'-O-CH3 modified nucleotide, and the nucleotide at position 9 is selected from a [2'-F-2'-F] modified nucleotide, a 2'-F modified nucleotide, or a 2'-O-CH3 modified nucleotide.

[0201] In some embodiments, starting from the 5' end, the nucleotides at positions 2, 6, and 12 of the antisense strand are 2'-F modified nucleotides, at least one nucleotide at position 9 is selected from [2'-F-2'-F] modified nucleotides, and the nucleotides at positions 14 and 16 are selected from 2'-F modified nucleotides or 2'-O-CH3 modified nucleotides. In some embodiments, starting from the 5' end, the nucleotides at positions 2, 6, and 12 of the antisense strand are 2'-F modified nucleotides, at least one nucleotide at positions 14 and 16 is selected from [2'-F-2'-F] modified nucleotides, and the nucleotide at position 9 is selected from 2'-F modified nucleotides or 2'-O-CH3 modified nucleotides.

[0202] In some embodiments, the 5th or 7th nucleotide of the antisense strand, starting from the 5' end, is selected from GNA or UNA. Preferably, the 7th nucleotide of the antisense strand, starting from the 5' end, is selected from GNA or UNA.

[0203] In some embodiments, the antisense strand has a 2'-O-MOE modified nucleotide, starting from the 5' end. In some embodiments, the 15th nucleotide of the antisense strand, starting from the 5' end, is a 2'-O-MOE modified nucleotide.

[0204] In some embodiments, starting from the 3' end, at least one of the nucleotides at positions 1 and 2 of the antisense strand is selected from a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me]. Preferably, starting from the 3' end, the nucleotide at position 1 of the antisense strand is selected from a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me], and the nucleotide at position 2 is a nucleotide modified with 2'-O-CH3. Preferably, starting from the 3' end, the nucleotide at position 2 of the antisense strand is selected from a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me], and the nucleotide at position 1 is a nucleotide modified with 2'-O-CH3. More preferably, starting from the 3' end, both the nucleotides at positions 1 and 2 of the antisense strand are selected from nucleotides modified with [2'-F-2'-Me] or nucleotides modified with [2'-OMe-2'-Me]. For example, starting from the 3' end, the first and second nucleotides of the antisense strand are both selected from nucleotides modified with [2'-F-2'-Me]; or, starting from the 3' end, the first and second nucleotides of the antisense strand are both selected from nucleotides modified with [2'-OMe-2'-Me]; or, starting from the 3' end, the first nucleotide of the antisense strand is selected from nucleotides modified with [2'-OMe-2'-Me], and the second nucleotide is selected from nucleotides modified with [2'-F-2'-Me]; or, starting from the 3' end, the first nucleotide of the antisense strand is selected from nucleotides modified with [2'-F-2'-Me], and the second nucleotide is selected from nucleotides modified with [2'-OMe-2'-Me].

[0205] In some embodiments, at least one of the nucleotides at positions 18 and 19 is selected from nucleotides modified with [2'-F-2'-Me] or nucleotides modified with [2'-OMe-2'-Me]; or, both of the nucleotides at positions 18 and 19 are selected from nucleotides modified with [2'-F-2'-Me] or nucleotides modified with [2'-OMe-2'-Me]. In some embodiments, at least one of the nucleotides at positions 20 and 21 is selected from nucleotides modified with [2'-F-2'-Me] or nucleotides modified with [2'-OMe-2'-Me]; or, both of the nucleotides at positions 20 and 21 are selected from nucleotides modified with [2'-F-2'-Me] or nucleotides modified with [2'-OMe-2'-Me].

[0206] Optionally, the antisense strand also has a phosphate thioester modification at one or more of the following positions: between the 1st and 2nd nucleotides starting from the 5' end, between the 2nd and 3rd nucleotides starting from the 5' end, between the 10th and 11th nucleotides starting from the 5' end, between the 1st and 2nd nucleotides starting from the 3' end, and / or between the 2nd and 3rd nucleotides starting from the 3' end.

[0207] Optionally, the antisense strand also has phosphate thioester modifications at the following positions: between the 1st and 2nd nucleotides starting from the 5' end, between the 2nd and 3rd nucleotides starting from the 5' end, between the 1st and 2nd nucleotides starting from the 3' end, and between the 2nd and 3rd nucleotides starting from the 3' end. Optionally, the antisense strand also has phosphate thioester modifications at the following positions: between the 1st and 2nd nucleotides starting from the 5' end, between the 2nd and 3rd nucleotides starting from the 5' end, between the 10th and 11th nucleotides starting from the 5' end, between the 1st and 2nd nucleotides starting from the 3' end, and between the 2nd and 3rd nucleotides starting from the 3' end. In some optional embodiments, the sense strand contains at least three of the following: a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, or a fourth modified nucleotide.

[0208] In some alternative embodiments, the positive strand contains a 2'-O-CH3 modified nucleotide and a 2'-F modified nucleotide, and in addition to the 2'-O-CH3 modified nucleotide and the 2'-F modified nucleotide, the positive strand also contains at least one of the following: a 2'-O-MOE modified nucleotide, a [2'-F-2'-F] modified nucleotide, a [2'-F-2'-Me] modified nucleotide, a [2'-OMe-2'-Me] modified nucleotide, LNA, CeNA, a 2'-deoxy modified nucleotide, or RNA (including phosphate thioester modified ribonucleotides). In some alternative embodiments, the positive strand contains a 2'-O-CH3 modified nucleotide and a 2'-F modified nucleotide, and in addition to the 2'-O-CH3 modified nucleotide and the 2'-F modified nucleotide, the positive strand also contains at least one of the following: a 2'-O-MOE modified nucleotide, a [2'-F-2'-F] modified nucleotide, a [2'-F-2'-Me] modified nucleotide, a [2'-OMe-2'-Me] modified nucleotide, LNA, CeNA, or a 2'-deoxy modified nucleotide.

[0209] In some optional embodiments, when the nucleotide on the positive strand contains at least one of a [2'-F-2'-F] modified nucleotide, a 2'-deoxy modified nucleotide, or RNA, it forms a base pair with any nucleotide at positions 10-13 and 15 of the antisense strand starting from the 5' end through complementary base pairing. In some optional embodiments, when the nucleotide on the positive strand contains at least one of a [2'-F-2'-F] modified nucleotide or a 2'-deoxy modified nucleotide, it forms a base pair with any nucleotide at positions 10-13 and 15 of the antisense strand starting from the 5' end through complementary base pairing. In some optional embodiments, when the nucleotide on the positive strand contains a [2'-F-2'-F] modified nucleotide, it forms a base pair with any nucleotide at positions 10-13 and 15 of the antisense strand starting from the 5' end through complementary base pairing.

[0210] In some optional embodiments, when the nucleotide on the positive strand contains a [2'-F-2'-Me] modified nucleotide or a [2'-OMe-2'-Me] modified nucleotide, it is located at the 3' end or 5' end of the positive strand.

[0211] In some alternative embodiments, when the nucleotide on the sense strand contains at least one of a 2'-O-MOE modified nucleotide, LNA, or cEt, it forms a base pair with any nucleotide at positions 1-9 and 14-19 of the antisense strand, starting from the 5' end, through complementary base pairing.

[0212] In some alternative embodiments, when the nucleotides on the sense strand contain at least 10 2'-O-methyl modified nucleotides, they form base pairs with any nucleotide at positions 1-10 and 14-19 of the antisense strand, starting from the 5' end, through complementary base pairing.

[0213] In some optional embodiments, when the nucleotides on the positive strand contain at least three 2'-F modified nucleotides, they form base pairs with any nucleotide at positions 10-13 and 15 of the antisense strand starting from the 5' end through complementary base pairing. In some optional embodiments, when the nucleotides on the positive strand contain three or four 2'-F modified nucleotides, they form base pairs with any nucleotide at positions 10-13 and 15 of the antisense strand starting from the 5' end through complementary base pairing.

[0214] In some alternative embodiments, when the nucleotides on the sense strand contain 3 or 4 2'-F modified nucleotides, they form base pairs with any nucleotide at positions 10-13 and 15 of the antisense strand starting from the 5' end through complementary base pairing, and the other nucleotides on the sense strand are selected from 2'-O-methyl modified nucleotides, LNA, or cEt.

[0215] In some alternative embodiments, when the nucleotides on the sense strand contain three or four 2'-F modified nucleotides, they form base pairs with any nucleotide at positions 10-13 and 15 of the antisense strand starting from the 5' end through complementary base pairing. The other nucleotides on the sense strand are selected from 2'-O-methyl modified nucleotides, LNA, or cEt, and when LNA or cEt is present, the sense strand contains only one LNA or cEt. Preferably, the LNA or cEt is located at position 1 starting from the 5' end.

[0216] Optionally, the positive strand also has a phosphate thioester modification at one or more of the following positions: between the first and second nucleotides starting from the 5' end, between the second and third nucleotides starting from the 5' end, between the first and second nucleotides starting from the 3' end, and / or between the second and third nucleotides starting from the 3' end. Preferably, the positive strand also has a phosphate thioester modification between the first and second nucleotides starting from the 5' end and between the second and third nucleotides starting from the 5' end; or, the positive strand also has a phosphate thioester modification between the first and second nucleotides starting from the 5' end, between the second and third nucleotides starting from the 5' end, and between the first and second nucleotides starting from the 3' end.

[0217] In some specific embodiments, the double-stranded oligonucleotide is selected from siRNA.

[0218] Conjugates:

[0219] In a second aspect, this disclosure provides conjugates comprising the double-stranded oligonucleotide of the first aspect or a pharmaceutically acceptable salt thereof, and one or more targeted delivery ligands.

[0220] In some alternative implementations, the targeted delivery ligand is selected from liver-targeted delivery ligands, kidney-targeted delivery ligands, lung-targeted delivery ligands, muscle-targeted delivery ligands, fat-targeted delivery ligands, heart-targeted delivery ligands, or central nervous system (CNS)-targeted delivery ligands.

[0221] In some alternative implementations, the liver-targeted delivery ligand is the GalNAc ligand.

[0222] In some alternative embodiments, the targeted delivery ligand conjugate is attached to the antisense strand and / or the sense strand. In other alternative embodiments, at least one of the 3' end of the sense strand, the 5' end of the sense strand, the 3' end of the antisense strand, and the 5' end of the antisense strand is conjugate to the targeted delivery ligand.

[0223] In some alternative implementations, the targeted delivery ligand is selected from one, two, three, or four.

[0224] In some alternative embodiments, there is one targeted delivery ligand. One of the targeted delivery ligands is conjugated to the 3' end of the sense strand, or the 5' end of the sense strand, or the 3' end of the antisense strand, or the 5' end of the antisense strand. In some specific embodiments, there is one targeted delivery ligand, and one of the targeted delivery ligands is conjugated to the 3' end of the sense strand.

[0225] In some specific embodiments, the targeted delivery ligand is selected from the structure of formula (CR01008)×3, its stereoisomer, its tautomer, or a pharmaceutically acceptable salt thereof. The structural formula of formula (CR01008)×3 is shown below:

[0226] In some specific embodiments, the targeted delivery ligand is selected from the structure of formula (CR01008)×4, its stereoisomers, its tautomers, or its pharmaceutically acceptable salts. The structural formula of formula (CR01008)×4 is shown below:

[0227] In some specific embodiments, the targeted delivery ligand is selected from the structure shown in Formula L96, its stereoisomers, its tautomers, or its pharmaceutically acceptable salts. The structural formula of Formula L96 is:

[0228] Composition:

[0229] Thirdly, this disclosure provides compositions comprising the double-stranded oligonucleotide of the first aspect or a pharmaceutically acceptable salt thereof and / or the conjugates of the second aspect.

[0230] In some specific embodiments, the composition comprises a pharmaceutically acceptable carrier or excipient.

[0231] use:

[0232] Fourthly, this disclosure provides the use of any of the following in the preparation of a medicament for the prevention and / or treatment of diseases:

[0233] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0234] (II) The conjugates described in the second aspect; and / or

[0235] (III) The composition described in the third aspect.

[0236] In some specific embodiments, the disease is a disease or symptom mediated by a target gene.

[0237] In some specific embodiments, the disease is a disease or symptom related to the expression of the target gene.

[0238] In some specific embodiments, the target gene is selected from CFB, C4B, ANGPTL3, SEAP, HBV-ETV, AGT, FTO, INHBE, USP20, URAT1, ANAPP, APOE4, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR or TTR, LRRK2, DUX4, complement 3, complement 5, NMDA, complement factor B, or RHO. In some embodiments, the target gene is SOD1, CFB, SEAP, C4B, or ANGPTL3.

[0239] In some specific embodiments, the disease is selected from conditions related to the aforementioned gene expression dysregulation.

[0240] Fifthly, this disclosure provides the use of any of the following in the preparation of a medicament for reducing the expression or activity of a target gene:

[0241] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0242] (II) The conjugates described in the second aspect; and / or

[0243] (III) The composition described in the third aspect.

[0244] In some specific embodiments, the target gene is selected from CFB, C4B, ANGPTL3, SEAP, HBV-ETV, AGT, FTO, INHBE, USP20, URAT1, ANAPP, APOE4, ATXN2, C9orf72, TARDBP, MAPT, HTT, SNCA, FUS, ATXN3, ATXN1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, RPTOR or TTR, LRRK2, DUX4, complement 3, complement 5, NMDA, complement factor B, or RHO. In some embodiments, the target gene is SOD1, CFB, SEAP, C4B, or ANGPTL3.

[0245] Pharmaceutical composition:

[0246] Sixthly, this disclosure provides pharmaceutical compositions comprising any one of the following and pharmaceutically acceptable excipients:

[0247] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0248] (II) The conjugates described in the second aspect; and / or

[0249] (III) The composition described in the third aspect.

[0250] method:

[0251] In a seventh aspect, this disclosure provides a method for preventing and / or treating a disease, said method comprising contacting cells with any of the following:

[0252] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0253] (II) The conjugates described in the second aspect; and / or

[0254] (III) The composition described in the third aspect; and / or

[0255] (IV) The pharmaceutical composition described in aspect 6.

[0256] In some alternative embodiments, the method of preventing and / or treating the disease includes contacting cells with an effective amount of the double-stranded oligonucleotide, its conjugate, composition, or pharmaceutical composition of the present disclosure. In some alternative embodiments, the method of preventing and / or treating the disease includes contacting cells with a therapeutically effective amount of the double-stranded oligonucleotide, its conjugate, composition, or pharmaceutical composition of the present disclosure.

[0257] In some embodiments, the cells are liver cells, kidney cells, lung cells, muscle cells, fat cells, heart cells, or central nervous system (CNS) cells.

[0258] In some embodiments, the cells are liver cells.

[0259] Eighthly, this disclosure provides a method for reducing the expression or activity of a target gene, said method comprising contacting a cell with any of the following:

[0260] (I) the double-stranded oligonucleotide described in the first aspect or a pharmaceutically acceptable salt thereof; and / or

[0261] (II) The conjugates described in the second aspect; and / or

[0262] (III) The composition described in the third aspect; and / or

[0263] (IV) The pharmaceutical composition described in aspect 6.

[0264] In some alternative embodiments, the method of reducing the expression or activity of the target gene includes contacting cells with an effective amount of the double-stranded oligonucleotide of the present disclosure or its pharmaceutically acceptable salts, conjugates, compositions, or pharmaceutical compositions. In some alternative embodiments, the method of reducing the expression or activity of the target gene includes contacting cells with a therapeutically effective amount of the double-stranded oligonucleotide of the present disclosure or its pharmaceutically acceptable salts, conjugates, compositions, or pharmaceutical compositions.

[0265] In some embodiments, the cells are liver cells, kidney cells, lung cells, muscle cells, fat cells, heart cells, or central nervous system (CNS) cells.

[0266] In some embodiments, the cells are liver cells.

[0267] In a ninth aspect, this disclosure provides a kit comprising the double-stranded oligonucleotide of the first aspect or a pharmaceutically acceptable salt thereof, the conjugate of the second aspect, the composition of the third aspect and / or the pharmaceutical composition of the sixth aspect; and instructions for use optionally for the prevention and / or treatment of disease or for reducing the expression or activity of a target gene.

[0268] In a tenth aspect, this disclosure provides the double-stranded oligonucleotides of the first aspect or pharmaceutically acceptable salts thereof for the prevention and / or treatment of diseases, the conjugates of the second aspect, the compositions of the third aspect, and / or the pharmaceutical compositions of the sixth aspect.

[0269] In the eleventh aspect, this disclosure provides the double-stranded oligonucleotides of the first aspect or pharmaceutically acceptable salts thereof for reducing the expression or activity of target genes, the conjugates of the second aspect, the compositions of the third aspect, and / or the pharmaceutical compositions of the sixth aspect.

[0270] In a twelfth aspect, this disclosure provides the use of the double-stranded oligonucleotides described in the first aspect or pharmaceutically acceptable salts thereof, the conjugates described in the second aspect, the compositions described in the third aspect, and / or the pharmaceutical compositions described in the sixth aspect for the prevention and / or treatment of diseases, or for reducing the expression or activity of target genes.

[0271] In some alternative embodiments, the double-stranded oligonucleotide of the first aspect or a pharmaceutically acceptable salt thereof, the conjugate of the second aspect, the composition of the third aspect, or the pharmaceutical composition of the sixth aspect are administered in a buffered or unbuffered solution.

[0272] In some alternative embodiments, inhibition of target gene expression means that target gene expression is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%. The inhibition rate of target gene expression can be determined by conventional methods known in the art, such as, but not limited to, luciferase reporter gene assay, PCR, or protein-based methods, such as immunofluorescence analysis, such as Western blotting or flow cytometry.

[0273] In some alternative embodiments, the above-described double-stranded oligonucleotide or its pharmaceutically acceptable salt, and / or the above-described conjugates are administered at doses of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg.

[0274] In some alternative embodiments, the above-described double-stranded oligonucleotide or its pharmaceutically acceptable salt, and / or the above-described conjugate, are administered subcutaneously or intravenously.

[0275] The content of this invention can be illustrated by way of example through the description in the following numbered paragraphs:

[0276] 1. A double-stranded oligonucleotide, characterized in that the double-stranded oligonucleotide comprises a sense strand and an antisense strand, each strand having 16 to 30 nucleotides, and the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region having 16 to 21 base pairs; the double-stranded oligonucleotide has at least one dangling end, and the dangling end is not located at the 5' end of the antisense strand.

[0277] The double-stranded region contains at least one [2'-R1-2'-R2] modified nucleotide;

[0278] R1 represents the substituent group of the 2'-OH ribose ring in the substituted nucleotide, and R2 represents the substituent group of the 2'-H ribose ring in the substituted nucleotide; R1 and R2 are each independently selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups.

[0279] 2. The double-stranded oligonucleotide according to paragraph 1, characterized in that, in the double-stranded region, at least one nucleotide selected from the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand (starting from the 5' end) and the nucleotides at positions 10-13 and 15 of the sense strand (starting from the 5' end) that are base-complementarily paired with the nucleotides at positions 10-13 and 15 of the antisense strand (starting from the 5' end) is selected from nucleotides modified with [2'-R1-2'-R2].

[0280] Optionally, the second nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0281] Optionally, the 6th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0282] Optionally, the 9th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0283] Optionally, the 14th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0284] Optionally, the 16th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2].

[0285] Optionally, the 14th and 16th nucleotides of the antisense strand, starting from the 5' end, are selected from nucleotides modified with [2'-R1-2'-R2].

[0286] Optionally, the nucleotide in the sense strand that pairs with the 10th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2].

[0287] Optionally, the nucleotide in the sense strand that pairs with the 11th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2].

[0288] Optionally, the nucleotide in the sense strand that pairs with the 12th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2].

[0289] Optionally, the nucleotide in the sense strand that pairs with the 13th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2].

[0290] Optionally, the nucleotide in the sense strand that is base-complementarily paired with the 15th nucleotide of the antisense strand starting from the 5' end is selected from nucleotides modified with [2'-R1-2'-R2].

[0291] 3. The double-stranded oligonucleotide according to paragraph 1 or 2, characterized in that at least five nucleotides from the 2nd, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand, starting from the 5' end, are each independently selected from nucleotides modified with [2'-R1-2'-R2] or nucleotides modified with 2'-fluorine, and the antisense strand contains no more than five nucleotides modified with 2'-fluorine; and / or, at least four nucleotides from the nucleotides that are base-complementary to the nucleotides at positions 10-13th and 15th positions of the antisense strand, starting from the 5' end, are each independently selected from nucleotides modified with [2'-R1-2'-R2] or nucleotides modified with 2'-fluorine, and the positive strand contains no more than four nucleotides modified with 2'-fluorine.

[0292] Furthermore, among the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand (starting from the 5' end) and the nucleotides at positions 10-13 and 15 of the sense strand (starting from the 5' end) that are base-complementary, at least one nucleotide is selected from a nucleotide modified with [2'-R1-2'-R2].

[0293] 4. The double-stranded oligonucleotide according to any one of paragraphs 1-3, characterized in that the second nucleotide of the antisense strand, starting from the 5' end, is selected from nucleotides modified with [2'-R1-2'-R2], and at least four nucleotides selected from the 6th, 9th, 12th, 14th, and 16th nucleotides are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0294] Optionally, the 6th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]; at least four of the nucleotides at positions 2, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0295] Optionally, the 14th nucleotide starting from the 5' end of the antisense strand is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 9, 12, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0296] Optionally, the 16th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 9, 12, and 14 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0297] Optionally, the 14th and 16th nucleotides of the antisense strand, starting from the 5' end, are selected from nucleotides modified with [2'-R1-2'-R2], and the nucleotides at positions 2, 6, 9, and 12 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0298] Optionally, the 9th nucleotide starting from the 5' end of the antisense strand is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

[0299] 5. The double-stranded oligonucleotide according to any one of paragraphs 1-4, characterized in that the nucleotide in the sense strand that pairs with the 10th nucleotide of the antisense strand by base complementarity starting from the 5' end is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the antisense strand that pair with the 11th-13th and 15th nucleotides of the antisense strand by base complementarity starting from the 5' end are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0300] Optionally, the nucleotide in the sense strand that pairs with the 11th nucleotide of the antisense strand starting from the 5' end via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th, 12th-13th, and 15th nucleotides of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0301] Optionally, the nucleotide in the sense strand that pairs with the 12th nucleotide of the antisense strand starting from the 5' end via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th-11th, 13th, and 15th nucleotides of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0302] Optionally, the nucleotide in the sense strand that pairs with the 13th nucleotide starting from the 5' end of the antisense strand via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th-12th and 15th nucleotides starting from the 5' end of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0303] Optionally, the nucleotide in the sense strand that pairs with the 15th nucleotide starting from the 5' end of the antisense strand via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th-13th nucleotides starting from the 5' end of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

[0304] 6. The double-stranded oligonucleotide according to any one of paragraphs 1-5, characterized in that R1 and R2 are each independently selected from halogens or optionally substituted C1-C6 alkoxy groups;

[0305] Optionally, R1 is selected from F or methoxy, and R2 is selected from F or methyl;

[0306] Optionally, the [2'-R1-2'-R2] modified nucleotide is selected from the [2'-F-2'-F] modified nucleotide, the [2'-F-2'-CH3] modified nucleotide, or the [2'-OCH3-2'-CH3] modified nucleotide.

[0307] 7. The double-stranded oligonucleotide according to paragraph 1, characterized in that the double-stranded oligonucleotide has at least one dangling end, the dangling end being composed of 1-3 modified nucleotides, and at least one [2'-R1-2'-R2] modified nucleotide is present in the dangling end;

[0308] Optionally, the double-stranded oligonucleotide has two dangling ends, and the dangling ends are located at the 3' end of the antisense strand and the 3' end of the sense strand, respectively.

[0309] Optionally, the double-stranded oligonucleotide has a dangling end, and the dangling end is located at the 3' end of the antisense strand;

[0310] Optionally, the double-stranded oligonucleotide has a dangling end, and the dangling end is located at the 3' end of the positive strand;

[0311] Optionally, the overhang is composed of 1-3 [2'-R1-2'-R2] modified nucleotides;

[0312] Optionally, the overhang is composed of two [2'-R1-2'-R2] modified nucleotides;

[0313] Optionally, the overhang is composed of two [2'-F-2'-CH3] modified nucleotides.

[0314] 8. The double-stranded oligonucleotide according to any one of paragraphs 1-7, characterized in that the double-stranded oligonucleotide contains at least four different modified nucleotides; wherein the double-stranded oligonucleotide includes a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, and a fourth modified nucleotide;

[0315] The first modified nucleotide is selected from nucleotides modified with [2'-R1-2'-R2].

[0316] The second modified nucleotide is selected from 2'-O-(CH2). n -R3 modified nucleotide; n is selected from integers from 0 to 3, and R3 is selected from optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, or... R 3a R 3b and R 3c Each is independently selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy;

[0317] The third modified nucleotide includes at least one of a 2'-F modified nucleotide or a 2'-deoxy modified nucleotide;

[0318] The fourth modified nucleotide includes Acyclic modified nucleotides, At least one of the following; wherein the acyclic modified nucleotides include At least one of them;

[0319] Where Base represents nucleobases A, U, G, C or T, and Z is independently selected from -OH or -SH;

[0320] R 4a and R 4bEach is independently selected from H or optionally substituted C1-C3 alkyl groups; j1 is selected from 1, 2 or 3;

[0321] R 5a and R 5b Each is independently selected from H or optionally substituted C1-C3 alkyl groups; j2 is selected from 1, 2 or 3; j3 is selected from 1, 2, 3 or 4; j4 is selected from 1, 2, 3 or 4;

[0322] R6 is selected from H or optionally substituted C1-C3 alkyl groups;

[0323] R7 is selected from H, optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl or optionally substituted C10-C22 alkynyl;

[0324] R8 is selected from Optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl, or optionally substituted C10-C22 alkynyl;

[0325] Optionally, the first modified nucleotide includes at least one of the following: a nucleotide modified with [2'-F-2'-F], a nucleotide modified with [2'-F-2'-CH3], or a nucleotide modified with [2'-OCH3-2'-CH3].

[0326] Optionally, the second modified nucleotide includes at least one of the following: a nucleotide modified with 2'-O-CH3, a nucleotide modified with 2'-O-CH2-O-CH2-CH3, a nucleotide modified with 2'-O-CH2-O-CH2-CF3, a nucleotide modified with 2'-O-CH2-CH2-O-CH3(2'-O-MOE), a nucleotide modified with 2'-O-TBDMS, a nucleotide modified with 2'-O-TIPS, or a nucleotide modified with 2'-O-TOM.

[0327] Optionally, the fourth modified nucleotide includes At least one of them;

[0328] Optionally, the fourth modified nucleotide includes at least one of LNA, CeNA, GNA, or UNA.

[0329] 9. The double-stranded oligonucleotide according to any one of paragraphs 1-8, characterized in that the double-stranded oligonucleotide further comprises a 5'-terminal phosphorylation modification, said 5'-terminal phosphorylation modification comprising 5'-(E)-vinylphosphonate (5'-(E)-VP) modification, 5'-methylphosphonate (5'-MP) modification, (S)-5'-C-methyl analog modification and 5'-thiophosphate (5'-PS) modification or analogs thereof.

[0330] 10. The double-stranded oligonucleotide according to paragraph 8 or 9, characterized in that the antisense strand comprises at least three of a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, or a fourth modified nucleotide;

[0331] Optionally, each nucleotide of the antisense strand is independently selected from [2'-F-2'-F] modified nucleotides, [2'-F-2'-Me] modified nucleotides, [2'-OMe-2'-Me] modified nucleotides, 2'-O-CH3 modified nucleotides, 2'-O-MOE modified nucleotides, 2'-F modified nucleotides, 2'-deoxy modified nucleotides, LNA, CeNA, or RNA;

[0332] Optionally, when the modification of the nucleotide on the antisense strand includes at least one of [2'-F-2'-F] modification, 2'-deoxy modification or RNA, it is located at at least one of the 2nd, 4th, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand starting from the 5' end;

[0333] Optionally, when the modification of the nucleotide on the antisense strand includes at least one of [2'-F-2'-Me] modification or [2'-OMe-2'-Me] modification, it is located at at least one of the 1st to 3rd positions of the antisense strand starting from the 3' end;

[0334] Optionally, when the modification of the nucleotide on the antisense strand includes at least one of 2'-O-MOE modification, LNA or CeNA, it is located at one or more positions of the 5th-10th, 15th, 17th-19th positions of the antisense strand starting from the 5' end;

[0335] Optionally, the modification of the nucleotide on the antisense strand is selected from at least 10 positions of the antisense strand, starting from the 5' end, when the 2'-O-methyl modification is performed;

[0336] Optionally, when the nucleotide modification on the antisense strand is selected from 2'-F modification, it is located at at least four of the 2nd, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand starting from the 5' end.

[0337] 11. The double-stranded oligonucleotide according to any one of paragraphs 8-10, characterized in that the antisense strand contains one or more of the following features:

[0338] 1) The first nucleotide of the antisense strand, starting from the 5' end, is selected from at least one of 5'-(E)-vinylphosphonate (5'-(E)-VP) modification, 5'-methylphosphonate (5'-MP) modification, (S)-5'-C-methyl analog, and 5'-thiophosphate (5'-PS) modification or analogs thereof; preferably, the first nucleotide of the antisense strand, starting from the 5' end, is selected from 5'-(E)-vinylphosphonate modification;

[0339] 2) The nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, include at least one of a [2'-F-2'-F] modified nucleotide, a 2'-deoxy modified nucleotide, or RNA, and the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, contain at least three 2'-F modified nucleotides; preferably, the nucleotide at position 14 of the antisense strand, starting from the 5' end, is a [2'-F-2'-F] modified nucleotide;

[0340] 3) The nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include the first modifying nucleotide, the second modifying nucleotide, and... Or at least one of the acyclic modified nucleotides; preferably, the nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of the following: 2'-O-MOE modified nucleotide, 2'-O-TBDMS modified nucleotide, [2'-F-2'-Me] modified nucleotide, [2'-OMe-2'-Me] modified nucleotide, GNA, UNA, LNA, or cEt, and the other nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from 2'-O-methyl modified nucleotides;

[0341] 4) At least one nucleotide at the 5th, 6th, and 7th positions of the antisense strand, starting from the 5' end, is selected from GNA or UNA, and the other nucleotides at the 5th, 6th, and 7th positions of the antisense strand, starting from the 5' end, are selected from nucleotides modified with 2'-O-CH3.

[0342] 5) The nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of the following: a nucleotide modified with 2'-O-MOE, a nucleotide modified with 2'-O-TBDMS, a nucleotide modified with [2'-OMe-2'-Me], or LNA, and the other nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from nucleotides modified with 2'-O-CH3;

[0343] 6) The nucleotides at positions 11-13 of the antisense strand, starting from the 5' end, are each independently selected from at least one of a nucleotide modified with 2'-O-CH3 or a nucleotide modified with 2'-F; preferably, the nucleotides at positions 11-13 of the antisense strand, starting from the 5' end, are all nucleotides modified with 2'-O-CH3, or the nucleotides at positions 11 and 13 of the antisense strand, starting from the 5' end, are selected from nucleotides modified with 2'-O-CH3, and the nucleotide at position 12 is selected from nucleotides modified with 2'-F.

[0344] 12. The double-stranded oligonucleotide according to any one of paragraphs 8-11, characterized in that the sense strand contains at least three of a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, or a fourth modified nucleotide;

[0345] Optionally, the positive strand contains a nucleotide modified with 2'-O-CH3 and a nucleotide modified with 2'-F, and in addition to the nucleotide modified with 2'-O-CH3 and the nucleotide modified with 2'-F, the positive strand also contains at least one of the following: a nucleotide modified with 2'-O-MOE, a nucleotide modified with [2'-F-2'-F], a nucleotide modified with [2'-F-2'-Me], a nucleotide modified with [2'-OMe-2'-Me], LNA, CeNA, DNA, or RNA.

[0346] Optionally, when the nucleotide on the sense strand contains at least one of a [2'-F-2'-F] modified nucleotide, a 2'-deoxy modified nucleotide, or RNA, it forms a base pair with any nucleotide at positions 10-13 and 15 of the antisense strand, starting from the 5' end, through complementary base pairing.

[0347] Optionally, when the nucleotide on the positive strand contains a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me], it is located at the 3' end or 5' end of the positive strand;

[0348] Optionally, when the nucleotide on the sense strand contains at least one of a 2'-O-MOE modified nucleotide, LNA, or cEt, it forms a base pair with any nucleotide at positions 1-9 and 14-19 of the antisense strand, starting from the 5' end, through complementary base pairing.

[0349] Optionally, when the nucleotides on the sense strand contain at least 10 nucleotides modified with 2'-O-methyl, they form base pairs with any nucleotides at positions 1-10 and 14-19 of the antisense strand, starting from the 5' end, through complementary base pairing.

[0350] Optionally, when the nucleotides on the sense strand contain at least three 2'-F modified nucleotides, they form base pairs with any nucleotide at positions 10-13 and 15 of the antisense strand, starting from the 5' end, through complementary base pairing.

[0351] 13. The double-stranded oligonucleotide according to any one of paragraphs 1-12, characterized in that the double-stranded oligonucleotide is selected from siRNA.

[0352] 14. A conjugate, characterized in that the conjugate comprises the double-stranded oligonucleotide described in any one of paragraphs 1-13 and one or more targeted delivery ligands;

[0353] Optionally, the targeted delivery ligand is selected from liver-targeted delivery ligands, kidney-targeted delivery ligands, lung-targeted delivery ligands, muscle-targeted delivery ligands, fat-targeted delivery ligands, heart-targeted delivery ligands, or central nervous system (CNS)-targeted delivery ligands;

[0354] Optionally, the liver-targeted delivery ligand is a GalNAc ligand.

[0355] 15. A composition, characterized in that the composition comprises a double-stranded oligonucleotide as described in any one of paragraphs 1-13 and / or a conjugate as described in paragraph 14.

[0356] 16. Any of the following uses in the preparation of medicaments for the prevention and / or treatment of diseases:

[0357] (I) Double-stranded oligonucleotides as described in any of paragraphs 1-13; and / or

[0358] (II) Conjugates as described in paragraph 14; and / or

[0359] (III) The composition as described in paragraph 15.

[0360] 17. The use of any of the following in the preparation of a drug for reducing the expression or activity of a target gene:

[0361] (I) Double-stranded oligonucleotides as described in any of paragraphs 1-13; and / or

[0362] (II) Conjugates as described in paragraph 14; and / or

[0363] (III) The composition as described in paragraph 15.

[0364] 18. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises any one of the following and a pharmaceutically acceptable excipient:

[0365] (I) Double-stranded oligonucleotides as described in any of paragraphs 1-13; and / or

[0366] (II) Conjugates as described in paragraph 14; and / or

[0367] (III) The composition as described in paragraph 15.

[0368] 19. A method for reducing the expression or activity of a target gene, characterized in that the method comprises contacting the cell with any of the following:

[0369] (I) Double-stranded oligonucleotides as described in any of paragraphs 1-13; and / or

[0370] (II) Conjugates as described in paragraph 14; and / or

[0371] (III) The composition as described in paragraph 15; and / or

[0372] (IV) Pharmaceutical compositions as described in paragraph 18.

[0373] Example

[0374] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0375] Unless otherwise stated, all siRNA sequences used in this disclosure were synthesized by Suzhou Beixin Biotechnology Co., Ltd.; all PCR primers used in this disclosure were synthesized by Beijing Qingke Biotechnology Co., Ltd.; and all C57BL / 6J mice used in this disclosure were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0376] Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available products. The main reagents and consumables are shown in Table 1, and the main instruments and equipment are shown in Table 2.

[0377] Table 1 Main Reagents and Consumables

[0378] Table 2 Main Instruments and Equipment

[0379] Preparation Example 1: Preparation of Compound NM127

[0380] In this preparation example, the synthetic route for compound NM127 is as follows:

[0381] (1-1) Synthesis of compound NM127-2

[0382] At room temperature, compound NM127-1 (2 g, 5.11 mmol, 1 eq, N-(9-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide, CB number CB99303990) was dissolved in pyridine (20 mL), cooled to 0 °C in an ice bath, and 4,4'-bismethoxytriphenylmethyl chloride (2.24 g, 6.64 mmol, 1.3 eq, abbreviated as DMTrCl, CAS number 40615-36-9) was added in portions. The reaction solution was stirred at 25 °C for 1 hour until the reaction was complete. The reaction mixture was quenched with methanol, the solvent was removed by rotary evaporation, diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated sodium chloride aqueous solution (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to give a yellow solid, compound NM127-2 (2.3 g, yield 64.97%). MS ESI (m / z) = 694.3 [M+H] + .

[0383] (1-2) Synthesis of compound NM127

[0384] Compound NM127-2 (2.3 g, 3.32 mmol, 1 eq) was repeatedly dried with acetonitrile (3 × 30 mL), dissolved in dichloromethane (25 mL), and a solution of 3-{[bis(diisopropylamino)phosphoryl]oxy}propionitrile (1.5 g, 4.98 mmol, 1.5 eq) dried with acetonitrile (3 × 10 mL) in dichloromethane (25 mL) was added. 1H-imidazolium-4,5-dianitrile (313.2 mg, 2.66 mmol, 0.8 eq) was added. The mixture was purged with nitrogen three times, and the reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere. After the reaction was complete, the mixture was diluted with 50 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 30 mL), the organic phases were combined, washed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution gradient, petroleum ether: ethyl acetate = 2:1) to give 2.6 g of white solid NM127, yield 87.83%. MS ESI (m / z) = 894.4 [M+H] + .

[0385] Preparation Example 2: Preparation of Compound NM128

[0386] In this preparation example, the synthetic route for compound NM128 is as follows:

[0387] (2-1) Synthesis of compound NM128-2

[0388] At room temperature, compound NM128-1 (3 g, 10.8 mmol, 1 eq, CAS No. 95058-80-3) was dissolved in pyridine (30 mL), cooled to 0 °C in an ice bath, and DMTrCl (4.75 g, 14.04 mmol, 1.3 eq) was added in portions. The reaction mixture was stirred at 25 °C for 1 hour. After the reaction was complete, methanol was added to quench the reaction mixture, the solvent was removed by rotary evaporation, and the mixture was diluted with ethyl acetate (50 mL). The mixture was washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution gradient: petroleum ether / ethyl acetate = 1 / 1) to give compound NM128-2 (3.6 g, yield 57.42%) as a yellow solid. MS ESI (m / z) = 581.2 [M+H] + .

[0389] (2-2) Synthesis of compound NM128

[0390] The compound NM128-2 (3.6 g, 6.21 mmol, 1 eq), dehydrated by acetonitrile (3 × 30 mL), was dissolved in dichloromethane (25 mL). A solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (2.80 g, 9.32 mmol, 1.5 eq), dehydrated by acetonitrile (3 × 20 mL), in dichloromethane (36 mL) was added. 1H-imidazolium-4,5-dianitronidazole (586.2 mg, 4.97 mmol, 0.8 eq) was added. The mixture was purged with nitrogen three times. The reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere until the reaction was complete. The reaction mixture was diluted with 50 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 30 mL), the organic phases were combined, washed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (elution: petroleum ether / ethyl acetate = 2 / 1) to give a white solid NM128 (3.4 g, yield 70.25%). MS ESI (m / z) = 781.3 [M+H] + .

[0391] Preparation Example 3: Preparation of Compound NM131

[0392] In this preparation example, the synthetic route for compound NM131 is as follows:

[0393] (3-1) Synthesis of compound NM131-2.

[0394] At room temperature, compound NM131-1 (2 g, 7.58 mmol, 1 eq, CAS No. 114248-23-6) was dissolved in pyridine (20 mL), cooled to 0 °C in an ice bath, and DMTrCl (3.33 g, 9.85 mmol, 1.3 eq) was added in portions. The reaction mixture was stirred at 25 °C for 1 hour until the reaction was complete. The reaction mixture was quenched with methanol, the solvent was removed by rotary evaporation, diluted with ethyl acetate (50 mL), washed with saturated ammonium chloride aqueous solution (2 × 30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 1, v / v) to give compound NM131-2 (2.5 g, yield 58.21%) as a yellow solid. MS ESI (m / z) = 567.3 [M+H] + .

[0395] (3-2) Synthesis of compound NM131

[0396] Compound NM131-2 (2.5 g, 4.41 mmol, 1 eq), which was dehydrated three times with acetonitrile (30 mL each time), was dissolved in dichloromethane (25 mL). A dichloromethane (25 mL) solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.99 g, 6.62 mmol, 1.5 eq), which was dehydrated three times with acetonitrile (30 mL each time), was added. 1H-imidazolium-4,5-dianitronidazole (416.3 mg, 3.53 mmol, 0.8 eq) was added. The mixture was purged with nitrogen three times. The reaction solution was stirred at 25 °C for 1 hour under a nitrogen atmosphere until the reaction was complete. The reaction mixture was diluted with 50 mL of saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (3 × 30 mL), and the organic phases were combined. The organic phases were washed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give a white solid, compound NM131 (2.86 g, yield 84.62%). MS ESI (m / z) = 767.4 [M+H] + .

[0397] Preparation Example 4: Synthesis of Compound NM054

[0398] In this preparation example, the synthetic route of compound NM054 is shown below:

[0399] (4-1) Synthesis of compound NM054-2

[0400] Compound NM054-1 (3 g, 11.54 mmol, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methylurea, CAS No. 863329-66-2) and pyridine (30 mL) were added to a 500 mL reaction vessel. The mixture was cooled to 0 °C, and 4,4'-bismethoxytriphenylmethylchloro (4.29 g, 12.7 mmol, 1.1 eq) was added in portions. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 3 hours under a nitrogen atmosphere. HPLC showed no starting material. After the reaction was complete, the reaction solution was concentrated, and purified with purified water (50 mL) and ethyl acetate (50 mL) for extraction. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM054-2 (2.7 g, yield 41.7%). MS ESI (m / z) = 563.0 [M+H] + .

[0401] (4-2) Synthesis of compound NM054

[0402] Compound NM054-2 (2.7 g, 4.8 mmol, 1.0 eq) was added to a 100 mL reaction vessel. Bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.74 g, 5.76 mmol, 1.2 eq) was added in portions, followed by 4,5-dicyanimidazole (0.45 g, 3.8 mmol, 0.8 eq, abbreviated as DCI, CAS number 1122-28-7) and dichloromethane (27 mL). The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 3 hours under a nitrogen atmosphere. After the reaction was complete, sodium bicarbonate aqueous solution (20 mL) was added to the reaction solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reversed-phase column chromatography (elution: acetonitrile / water = 90 / 10, v / v) to obtain compound NM054 (3.0 g). MS ESI(m / z) = 763[M+H] + .

[0403] Preparation Example 5: Synthesis of Compound NM118

[0404] In this preparation example, the synthetic route of compound NM118 is as follows:

[0405] (5-1) Synthesis of compound NM118-2

[0406] Compound NM118-1 (20 g, 1.0 eq, 2'-C-methyluridine, CAS No. 31448-54-1) was dissolved in pyridine (100 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (28.6 g, 1.2 eq, TiPDSCl2, CAS No. 69304-37-6) was added. The mixture was stirred at room temperature for 12 hours until the reaction was complete. The reaction solution was concentrated, and water (100 mL) was added. The mixture was extracted twice with ethyl acetate (100 mL each time). The organic phase was dried and concentrated, and purified by normal phase (elution: ethyl acetate / petroleum ether = 34 / 66, v / v) to give compound NM118-2 (39 g) as a white solid. MS ESI (m / z) = 501 [M+H] + .

[0407] (5-2) Synthesis of compound NM118-3

[0408] Compound NM118-2 (5.0 g, 1 eq) was dissolved in anhydrous tetrahydrofuran (50 mL). A 60 wt% NaH solution (1.6 g, 4 eq) in tetrahydrofuran was added under ice bath conditions. The mixture was purged with nitrogen three times and stirred under ice bath conditions for 1 hour. Iodomethane (3 g, 2 eq) was then added, and the reaction was carried out at room temperature for 4 hours until completion. A saturated ammonium chloride aqueous solution (20 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (20 mL each time). The organic phase was dried and concentrated, and purified by reverse-phase chromatography (elution: acetonitrile / water = 25 / 75, v / v) to obtain a white powder, compound NM118-3 (3 g). MS ESI (m / z) = 515 [M+H] + .

[0409] (5-3) Synthesis of compound NM118-4

[0410] Compound NM118-3 (3.0 g, 1 eq) was dissolved in tetrahydrofuran (30 mL), and a 1 M tetrabutylammonium fluoride solution in tetrahydrofuran (3 mL, 0.5 eq) was added. The mixture was stirred at room temperature for 1 hour until the reaction was complete. Water (20 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (20 mL each time). The organic phase was dried and concentrated, and then purified by reverse-phase extraction (elution: acetonitrile / water = 36 / 64, v / v) to give compound NM118-4 (1.1 g) as a white powder. MS ESI (m / z) = 295 [M + Na] + .

[0411] (5-4) Synthesis of compound NM118-5

[0412] Compound NM118-4 (1.1 g, 1.0 eq) was dissolved in pyridine (20 mL). 4,4'-bismethoxytriphenylmethyl chloride (1.7 g, 1.3 eq) was added under ice bath conditions. The mixture was purged with nitrogen three times, stirred at room temperature for 3 hours, and then quenched with methanol (20 mL). The reaction was complete. The reaction solution was concentrated, and water (20 mL) was added. The mixture was extracted three times with ethyl acetate (20 mL each time). The organic phase was dried and concentrated, and purified by normal phase purification (elution: ethyl acetate / petroleum ether = 50 / 50, V / V) to give compound NM118-5 (1.8 g) as a pale yellow solid. MS ESI (m / z) = 575 [M+H] + .

[0413] (5-5) Synthesis of compound NM118

[0414] Compound NM118-5 (1.6 g, 1.0 eq) was dissolved in anhydrous dichloromethane (20 mL), and 4,5-dicyanimidazole (264.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.92 g, 1.1 eq) were added separately. The mixture was purged with nitrogen three times, and stirred at room temperature for 2 hours until the reaction was complete. A saturated aqueous solution of sodium bicarbonate (20 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 mL each time). The organic phase was dried and concentrated, and purified by reverse-phase chromatography (elution: acetonitrile / water = 75 / 25, v / v) to give a white powder, compound NM118 (1.7 g, yield 78.7%). MS ESI (m / z) = 775 [M+H] + .

[0415] 1 H NMR(400MHz, DMSO-d6)δ11.47(s,1H),7.99(dd,J=8.1,1.6Hz,1H),7.44–7.18(m,9H),6.92(ddd, J=10.7,7.4,3.5Hz,4H),5.96(d,J=9.5Hz,1H),5.77(s,1H),4.97(t,J=8.7Hz,1H),4.38–4.07(m, 2H),3.76(d,J=3.1Hz,7H),3.50(ddt,J=13.9,11.6,7.1Hz,4H),3.40(d,J=6.7Hz,3H),2.81(t,J =5.8Hz,1H),2.64–2.54(m,1H),1.25(d,J=3.3Hz,3H),1.19–1.03(m,10H),0.92(d,J=6.7Hz,2H).

[0416] Preparation Example 6: Synthesis of Compound CR01008 and Compound CR01008Z

[0417] In this preparation example, the synthetic routes for compounds CR01008 and CR01008Z are as follows:

[0418] (6-1) Synthesis of compound CR01008-2

[0419] Compound CR01008-1 (10.0 g, 1.0 eq, trans-4-(Boc-amino)cyclohexylformaldehyde, CAS No. 181308-57-6) and a 37 wt% formaldehyde aqueous solution (8.9 g, 2.4 eq) were dissolved in methanol (33 mL). A 45.3 wt% KOH aqueous solution (13 mL) was added dropwise. After the addition was complete, the mixture was stirred at 25 °C for 30 minutes, then heated to 60 °C and refluxed at 60 °C for 2 hours. The reaction was then complete. After the reaction solution cooled to room temperature, it was evaporated under reduced pressure to obtain a white solid crude product. A small amount of water was added to the crude product, and the mixture was slurried and filtered to obtain a white solid compound CR01008-2 (9 g, yield 78.9%). MS-ESI (m / z) = 260 [M+H] + .

[0420] (6-2) Synthesis of compound CR01008-3

[0421] Compound CR01008-2 (9 g, 1 eq) was dissolved in 1,4-dioxane (70 mL), and a 4 M solution of 1,4-dioxane hydrogen chloride (45 mL) was added. The mixture was stirred at 25 °C for 1 hour until the reaction was complete. The reaction solution was evaporated under reduced pressure to obtain a white solid compound CR01008-3 (6.8 g, 100% yield).

[0422] (6-3) Synthesis of compound CR01008-5

[0423] Compounds CR01008-3 (1.8 g, 2.0 eq), CR01008-4 (2.1 g, 1.0 eq, 5-[[(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]valeric acid, CAS No. 1159408-54-4), and N,N-diisopropylethylamine (3.5 g, 6.0 eq, abbreviated as DIEA, CAS No. 7087-68-5) were dissolved in DMF (N,N-dimethylformamide, 15 mL). Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (1.9 g, 1.1 eq, abbreviated as HBTU, CAS No. 94790-37-1) was added. The reaction system was stirred at 25 °C for 3 hours under a nitrogen atmosphere until the reaction was completed. The reaction mixture was evaporated to dryness under reduced pressure, and purified by reverse-phase extraction (elution buffer: acetonitrile / water = 22 / 78, v / v) to give a white solid, CR01008-5 (1.78 g, yield 64.4%). MS-ESI (m / z) = 589 [M+H] + .

[0424] (6-4) Synthesis of compound CR01008-6

[0425] Compound CR01008-5 (1.54 g, 1.0 eq) was dissolved in pyridine (15 mL). The reaction system was cooled to 0 °C using an ice-water bath, and DMTrCl (1.32 g, 1.5 eq) was added at 0 °C. The reaction was carried out at 25 °C for 3 hours, and then quenched with methanol (15 mL). The reaction was complete. The reaction solution was evaporated to dryness under reduced pressure, and purified by reverse-phase chromatography (elution: acetonitrile / water = 60 / 40, v / v) to give compound CR01008-6 (1 g, yield 42.7%) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0426] (6-5) Synthesis of compound CR01008

[0427] Compound CR01008-6 (1.08 g, 1.0 eq) was dissolved in anhydrous dichloromethane (20 mL). 4,5-Dicyanoimidazole (115 mg, 0.8 eq, abbreviated as DCI, CAS No. 1122-28-7) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (732 mg, 2.1 eq, CAS No. 102691-36-1) were added separately. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C for 2 hours under a nitrogen atmosphere until the reaction was complete. A saturated sodium bicarbonate aqueous solution (20 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (20 mL each time). The organic phases were combined, dried, and purified by reverse-phase extraction (elution: acetonitrile / water = 72 / 28, v / v) to obtain a white powder, compound CR01008 (1 g, yield 76.0%). MS-ESI(m / z) = 1091[M+Na] + .

[0428] 1H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37–1.17(m,5H),1.60–1.40(m,6H),1.68–1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58– 3.39(m,3H),3.69–3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93–6.87(m,4H),7.35–7.21(m,7H),7.44–7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0429] (6-6) Synthesis of compound CR01008-7

[0430] Compound CR01008-6 (500 mg) was dissolved in dichloromethane (10 mL), and succinic anhydride (112 mg), 4-dimethylaminopyridine (6.8 mg, abbreviated as DMAP, CAS No. 1122-58-3), and triethylamine (226.2 mg) were added. The mixture was purged with nitrogen three times, and the reaction system was stirred at 25 °C under a nitrogen atmosphere for 16 hours until the reaction was complete. The reaction solution was purified by FLASH to give compound CR01008-7 (300 mg, yield 53.6%). MS-ESI (m / z) = 10¹³ [M + Na] + .

[0431] (6-7) Synthesis of compound CR01008Z

[0432] Compound CR01008-7 (50 mg), aminoCPG (1.25 g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) were added to a 20 mL sample vial and reacted on a shaker for 16 hours until the reaction was complete. The reaction solution was filtered, and the filter cake was washed once with acetonitrile (10 mL) and then dried under vacuum. The dried filter cake, DMAP (3 mg), Cap1 (10 mL), and Cap2 (1 mL) were added to a 20 mL sample vial and reacted on a shaker for 6 hours until the reaction was complete. The reaction solution was filtered, and the filter cake was washed once with 10 mL of acetonitrile and then dried under vacuum to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0433] The structural formula of amino-CPG (Aminoalkyl-CPG) is as follows: Model C3006-1000, 100-200 mesh, loading 80μmol / g.

[0434] Cap1 and Cap2 are capping reagents. Cap1 is a 20 vol% N-methylimidazolium pyridine / acetonitrile mixed solution with a pyridine to acetonitrile volume ratio of 3:5. Cap2 is a 20 vol% acetic anhydride acetonitrile solution.

[0435] Preparation Example 7: Preparation of siRNA conjugates

[0436] Compound L96-PS was purchased from Asymchem Laboratories (Tianjin) Co., Ltd., with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is as follows:

[0437] PS represents a polystyrene resin solid support, wherein the hydroxyl group of N-acetylgalactosamine has an Ac protecting group.

[0438] The structural formula of compound NM130 is: Obtained through market purchase.

[0439] (7-1) Synthesis of the sense strand (SS) and antisense strand (AS) of the delivery vector, joined by 3' terminal ligation of the sense strand.

[0440] The phosphoramide solid-phase synthesis method for nucleic acids utilizes the aforementioned compounds linked to a solid-phase support (e.g., CPG support, PS support, compound L96-PS, compound CR01008Z) as the starting cycle, and nucleoside monomers are linked one by one according to the nucleotide sequence in the 3'-5' direction. During the synthesis, compounds NM127, NM128, NM130, NM131, NM054, NM118, and CR01008 are each considered as a nucleoside monomer.

[0441] Each connection of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfidation. The synthetic conditions are given below:

[0442] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0443] The deprotection reaction conditions were the same for each step. The deprotection reaction conditions were: temperature 25℃, reaction time 70 seconds, deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% by volume), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0444] 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).

[0445] The conditions for each capping reaction were identical. The conditions for the capping reaction were: temperature 25℃; reaction time 2 minutes; the capping reagent solution was a 1:1 molar ratio of Cap1 and Cap2, Cap1 being a 20 vol% N-methylimidazole pyridine / acetonitrile mixture with a pyridine to acetonitrile volume ratio of 3:5, and Cap2 being a 20 vol% 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.

[0446] The conditions for each oxidation / sulfidation reaction were identical. The oxidation reaction conditions were: temperature 25°C; reaction time 3 seconds; oxidizing agent concentration of 0.05M iodine solution; 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 (volume ratio of water to pyridine of 1:9). The sulfidation reaction conditions were: temperature 25°C; reaction time 360 ​​seconds; thioreagent concentration of 0.2M hydroflavin in pyridine solution; 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 (volume ratio of water to pyridine of 1:9).

[0447] 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:

[0448] The cleavage and deprotection conditions were as follows: The synthesized nucleotide sequence linked to a solid-phase support was added to 25% (w / w) 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.

[0449] 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 dextran gel) and elution with deionized water.

[0450] 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 sense strand and the target antisense strand had been obtained.

[0451] (7-2) Synthesis of siRNA conjugates

[0452] The sense and antisense strands synthesized in step (7-1) 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 siRNA conjugates with sense and antisense strands as shown in Tables 3-1 and 3-2.

[0453] When the delivery vector is L96, the structural formula of the siRNA conjugate is:

[0454] When the delivery vector is a tri-cluster CR01008 (denoted as: 5'-(CR01008)(CR01008)(CR01008Z)-3' or (CR01008)×3), the structural formula of the siRNA conjugate is:

[0455] When the delivery vector is a tetracluster of CR01008 (denoted as: 5'-(CR01008)(CR01008)(CR01008)(CR01008Z)-3' or (CR01008)×4), the structural formula of the siRNA conjugate is:

[0456] in, This indicates siRNA.

[0457] Table 3 Sequence information of siRNA in this application

[0458] Table 3-1 Sequence information of siRNA and its conjugates

[0459] Table 3-2 Sequence information of siRNA conjugates

[0460] Unless otherwise stated, 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 a nucleotide; lowercase letter m indicates that the nucleotide to its left adjacent uppercase letter is modified with 2'-O-CH3; lowercase letter f indicates that the nucleotide to its left adjacent uppercase letter is modified with 2'-F; (moe) indicates that the nucleotide to its left adjacent uppercase letter is modified with 2'-O-MOE; (LNA) indicates that the nucleotide to its left adjacent uppercase letter is LNA; (cEt) indicates that the nucleotide to its left adjacent uppercase letter is cEt; (GNA) indicates that the nucleotide to its left adjacent uppercase letter is GNA; (UNA) indicates that the nucleotide to its left adjacent uppercase letter is UNA; lowercase letter s indicates that the nucleoside bond between two adjacent nucleotides on its left and right sides is a phosphate thioester bond.

[0461] (NM127), (NM128), (NM129), (NM130), (NM131), (NM054) and (NM118) each represent a nucleotide.

[0462] in:

[0463] The structural formula of (NM127) is:

[0464] The structural formula of (NM128) is:

[0465] The structural formula of (NM129) is:

[0466] The structural formula of (NM130) is:

[0467] The structural formula of (NM131) is:

[0468] The structural formula of (NM054) is:

[0469] The structural formula of (NM118) is:

[0470] (LNA) indicates that the nucleotide represented by the capital letter on its left is a nucleotide with... Modified nucleotides, where Base represents the corresponding base;

[0471] (cEt) indicates that the nucleotide represented by the uppercase letter on the left is a nucleotide with... Modified nucleotides, where Base represents the corresponding base;

[0472] (GNA) indicates that the nucleotide represented by the capital letter on the left is a nucleotide with... Modified nucleotides, where Base represents the corresponding base;

[0473] (UNA) indicates that the nucleotide represented by the capital letter on the left is a nucleotide with... Modified nucleotides, where Base represents the corresponding base;

[0474] The structures of (CR01008)×3, (CR01008)×4, and L96 are shown above.

[0475] Table 4. Molecular weight information of siRNA conjugates

[0476] Biological detection experiments

[0477] Unless otherwise stated, the experimental animals C57BL / 6J mice used in this disclosure were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0478] Unless otherwise stated, all reagents, consumables, and instruments used in this disclosure are commercially available. The main reagents and consumables are shown in Table A, and the main instruments are shown in Table B.

[0479] Table A: Main Reagents and Consumables

[0480] Table B: Main Instruments and Equipment

[0481] Unless otherwise stated, the "method for evaluating the inhibitory activity of siRNA conjugates against target genes in primary mouse liver cells" used in this disclosure is as follows:

[0482] Primary hepatocyte isolation from mice: Primary hepatocytes were extracted from fresh liver tissue of C56BL / 6j mice. The specific procedure involved anesthetizing the mice with an intraperitoneal injection of a 10wt% chloral hydrate aqueous solution, fixing the mice, and disinfecting their abdomen and chest with a 75% (v / v) ethanol aqueous solution. Surgical instruments were sterilized, and the abdominal cavity was opened to expose the portal vein and inferior vena cava. A heparin cap was attached to an indwelling needle, which was then connected to a scalp vein needle attached to an IV drip bag (containing perfusion fluid, specifically 0.5 mM EDTA (CAS No. 60-00-4) in HBSS (Hank's Balanced Salt Solution)). The needle was inserted through the inferior vena cava, and perfusion was initiated at a rate of 120 drops / min. The portal vein was then cut to allow the perfusion fluid to drain out, and perfusion continued for 4 minutes. The perfusion fluid was then replaced (HBSS solution containing 0.8 mg / mL type IV collagenase (Sigma, C5138) (containing 0.08% DN1 enzyme (Sigma, DN25))) and perfusion continued for another 8 minutes. The perfused liver was removed from the animal and treated with HBSS (containing Ca... 2+ Mg 2+ The liver was cleaned with MACGENE (CC016); the liver was placed in a sterile culture dish and DMEM complete medium (DMEM medium + 10% serum) was added; the liver was shredded and the cell suspension was filtered through a cell sieve to remove undigested tissue and connective tissue, centrifuged at 800 rpm for 3 min and the supernatant was discarded; DMEM complete medium was added again and the suspension was centrifuged again to obtain primary mouse hepatocytes.

[0483] Cell culture and transfection: Adjust cell density to 2×10⁶ cells / year by adding DMEM complete medium. 5 Primary mouse hepatocyte suspension was obtained by measuring cells / mL. The cells were then seeded into 12-well culture plates pre-coated with type I rat tail collagen (coating method according to SolarBio (C8062) instructions at 2 μg / mL). 2 (Concentration coating), the volume of cell suspension added was 1000 μL / well, i.e., the cell quantity was 2 × 10⁻⁶. 5 Cells / well

[0484] Dilute each sequence group with PBS buffer to a final experimental concentration 1000 times (based on siRNA). Add 1 μL / well of the siRNA sequence working solution to each of the above 12-well culture plates, with 2-3 wells for each siRNA sequence. Add 1 μL / well of PBS buffer to the remaining 2-3 wells as blank control wells. Shake the culture plate to mix thoroughly. Incubate the culture plate in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0485] RNA extraction: Total RNA was extracted from each group of primary hepatocyte samples using a fully automated nucleic acid extractor and nucleic acid extraction kit from Zhejiang Hanwei Technology Co., Ltd., following the instructions.

[0486] Unless otherwise stated, the "method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice" used in this disclosure is as follows:

[0487] Six- to eight-week-old C57BL / 6J mice (all female) were randomly divided into groups based on body weight. The drug dosage for each group was calculated based on body weight and administered via a single subcutaneous injection. The experimental group received a solution containing the siRNA sequence, specifically prepared with PBS solution to the appropriate concentration (based on siRNA), at a dose of 5 mL / kg (mice). The PBS control group received a PBS solution without the drug sequence, at a dose of 5 mL / kg (mice). The day of administration was designated as day 0 (D0). At a predetermined time after administration, five mice from each group were sacrificed. The sacrificed mice were grossly dissected, and liver tissue was collected from each mouse. The liver tissue was cut into pieces approximately 2 mm thick. 3 Small pieces, stored using RNA Later.

[0488] Liver tissue samples were taken from different experimental groups at different time points from the RNA later sample. The liver tissue samples were homogenized in a Tissuelyser II fully automated tissue homogenizer for 60 seconds, and then total RNA was extracted using a fully automated nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd.) according to the standard operating procedure for total RNA extraction.

[0489] Unless otherwise stated, the "method for detecting mRNA expression levels" used in this disclosure is as follows:

[0490] 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). TMSelect Master Mix (Catalog number: 4472908) was used to detect the expression level of the target gene mRNA. 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. A 20 μL Real-time PCR reaction system was prepared for each PCR detection well according to the instructions of the real-time quantitative PCR kit. Each reaction system contained 5 μL of cDNA solution obtained from the above reverse transcription reaction and 10 μL of SYBR Green. TM Select Master Mix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 4 μL of Nase-Free H2O. Place the prepared reaction mixture in a real-time quantitative PCR instrument (ABI, StepOnePlus). TM Real-time PCR amplification was performed using a three-step method. The amplification program was 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. The denaturation, annealing, and extension process was repeated for 40 cycles.

[0491] Alternatively, a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used with Oligo(dT)18 reverse transcription primers. A 20 μL reverse transcription system was prepared according to the kit's instructions, and the reverse transcription reaction was completed. Then, a real-time quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) was used on a quantitative PCR instrument (Bio-Rad, CFX Opus 384) to detect the expression level of the target gene mRNA. In this real-time quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene. Primers targeting the target gene and primers targeting the GAPDH internal control gene were used to detect the target gene and the GAPDH internal control gene, respectively.

[0492] In this real-time quantitative PCR method, the ΔΔCt method was used to calculate the relative quantitative levels and inhibition rates of target gene mRNA in each test group. The calculation method is as follows:

[0493] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group);

[0494] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group);

[0495] ΔCt(test group) = ΔCt(test group) – ΔCt(control group average);

[0496] ΔCt(control group) = ΔCt(control group) – ΔCt(control group average).

[0497] In cell experiments, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) values ​​from several replicates in the control group. In animal experiments, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) values ​​from each of the five mice sacrificed at the same time point in the control group. Therefore, each sample in both the test and control groups corresponds to a ΔCt value.

[0498] The relative expression level of the target gene mRNA in the test group was 2. -ΔΔCt( (Test group) × 100%.

[0499] Using the control group as a baseline, the expression level of the target gene mRNA in the test group was normalized, and the expression level of the target gene mRNA in the control group was defined as 100%.

[0500] The inhibition rate of target gene mRNA expression in the test group (%) = (1 – relative expression level of target gene mRNA in the test group) × 100%.

[0501] Unless otherwise stated, all activity test data are expressed in terms of... It is noted that all experimental data were plotted and analyzed using GraphPad Prism 8.0 software.

[0502] Example 1: Evaluation of the inhibitory activity of an antisense siRNA conjugate containing (NM130) on the target gene complement factor B (CFB) in primary mouse hepatocytes.

[0503] Example 1 uses the "Method for evaluating the inhibitory activity of siRNA conjugates against target genes in primary mouse hepatocytes" to evaluate the inhibitory activity of the antisense strand conjugates RZm11503, RZm11504, RZm11505, RZm11506 and RZm11507 containing (NM130) and the reference conjugates RZm11001, RZm11501 and RZm11502 without (NM130) against the target gene CFB in primary mouse hepatocytes.

[0504] In contrast to the reference conjugate RZm11001, the antisense strand in conjugate RZm11503 is replaced by (NM130) at the 14th nucleotide starting from the 5' end.

[0505] Compared to the reference conjugate RZm11501, in conjugate RZm11504, the 14th nucleotide starting from the 5' end of the antisense strand is replaced with (NM130); in conjugate RZm11505, the 16th nucleotide starting from the 5' end of the antisense strand is replaced with (NM130); and in conjugate RZm11506, both the 14th and 16th nucleotides starting from the 5' end of the antisense strand are replaced with (NM130).

[0506] Compared to the reference conjugate RZm11502, the antisense strand in conjugate RZm11507 is replaced with (NM130) at the 14th nucleotide starting from the 5' end.

[0507] Primary mouse hepatocytes with a cell mass of 2 × 10⁻⁶ 5 Cells were seeded per well in a 12-well plate. The double-stranded siRNA conjugates for each group were serially diluted with PBS to a working solution of 10 μM (based on siRNA). 1 μL of the siRNA conjugate working solution was added to each well of the 12-well plate, equivalent to a final transfection concentration of 10 nM (based on siRNA). Two wells were used for each concentration of siRNA conjugate. Two additional wells were used as blank control wells, each containing 1 μL of PBS.

[0508] Table 5 Primer sequence information in Example 1

[0509] The results of Example 1 show that, compared with the reference conjugate without (NM130), the antisense strand containing (NM130) has a comparable or stronger target gene repression effect (Figure 1, Table 6).

[0510] Table 6. Inhibitory activity of siRNA conjugate on target gene CFB in primary mouse liver cells.

[0511] Example 2: Evaluation of the inhibitory activity of a siRNA conjugate containing (NM130) in the sense strand against the target gene CFB in primary mouse liver cells.

[0512] Example 2 used the "Method for evaluating the inhibitory activity of siRNA conjugates against target genes in mouse primary liver cells" to evaluate the inhibitory activity of the positive strand conjugates RZm11510 and RZm11511 containing (NM130) and the reference conjugate RZm11015 without (NM130) against the target gene CFB in mouse primary liver cells.

[0513] Compared to the reference conjugate RZm11015, the 7th nucleotide of the positive strand in conjugate RZm11510 is replaced by (NM130) starting from the 5' end, and the 8th nucleotide of the positive strand in conjugate RZm11511 is replaced by (NM130) starting from the 5' end.

[0514] Primary mouse hepatocytes with a cell mass of 2 × 10⁻⁶ 5 Cells were seeded per well in a 12-well plate. The double-stranded siRNA conjugates for each group were serially diluted to 10 μM (based on siRNA) with PBS solution. 1 μL of the siRNA conjugate working solution was added to each well of the 12-well plate, equivalent to a final transfection concentration of 10 nM (based on siRNA). Two wells were used for each siRNA concentration. Two additional wells were used as blank control wells, each containing 1 μL of PBS solution.

[0515] RNA extraction, reverse transcription, and quantitative real-time PCR were performed as described above. Gene expression differences were calculated using the ΔΔCt method. Primers are shown in Table 5 of Example 1.

[0516] The results of Example 2 show that, compared with the reference conjugate RZm11015 which does not contain (NM130), the conjugates RZm11510 and RZm11511 containing (NM130) in the positive chain have comparable or better inhibitory activity (Figure 2, Table 7).

[0517] Table 7. Inhibitory activity of siRNA conjugate on target gene CFB in primary mouse liver cells.

[0518] Example 3 evaluates the inhibitory activity of a siRNA conjugate containing (NM130) in the sense strand against the target gene superoxide dismutase 1 (SOD1) in primary mouse liver cells.

[0519] Example 3 used the "Method for evaluating the inhibitory activity of siRNA conjugates against target genes in mouse primary liver cells" to evaluate the inhibitory activity of the positive strand conjugates RZ599055, RZ599057 and RZ599056 containing (NM130) and the reference conjugate RZ599001 without (NM130) against the target gene SOD1 in mouse primary liver cells.

[0520] Compared to the reference conjugate RZ599001, in conjugates RZ599055 and RZ599057, the 8th nucleotide starting from the 5' end of the positive strand is replaced with (NM130), and in conjugate RZ599056, the 9th nucleotide starting from the 5' end of the positive strand is replaced with (NM130).

[0521] Primary mouse hepatocytes with a cell mass of 2 × 10⁻⁶ 5 Cells were seeded per well in a 12-well plate. The double-stranded siRNA conjugates for each group were serially diluted with PBS to 5 μM working solution (based on siRNA). 1 μL of the siRNA conjugate working solution was added to each well of the 12-well plate, equivalent to a final transfection concentration of 5 nM (based on siRNA). Two wells were used for each siRNA concentration. Two additional wells were used as blank control wells, each containing 1 μL of PBS.

[0522] RNA extraction, reverse transcription reaction, and quantitative real-time PCR were performed as described above. Gene expression differences were calculated using the ΔΔCt method.

[0523] Table 8 Primer sequence information in Example 3

[0524] The results of Example 3 show that, compared with the reference conjugate RZ599001, the conjugates RZ599055, RZ599056, and RZ599057 containing (NM130) in the sense chain have comparable or better inhibitory activity (Figure 3, Table 9).

[0525] Table 9. Inhibitory activity of siRNA conjugate on target gene SOD1 in primary mouse liver cells.

[0526] Example 4 evaluates the inhibitory activity of a siRNA conjugate containing (NM130) in the sense strand against the target gene angiopoietin-like 3 (ANGPTL3) in primary mouse hepatocytes.

[0527] Example 4 uses the "Method for evaluating the inhibitory activity of siRNA conjugates against target genes in mouse primary liver cells" to evaluate the inhibitory activity of the positive strand conjugate RZ597101 containing (NM130) and the reference conjugate RZ597002 without (NM130) against the target gene ANGPTL3 in mouse primary liver cells.

[0528] Compared to the reference conjugate RZ597002, the positive strand of conjugate RZ597101 is replaced with (NM130) at the 8th nucleotide starting from the 5' end.

[0529] Primary mouse hepatocytes with a cell mass of 2 × 10⁻⁶ 5Cells / well were seeded into 12-well culture plates. The double-stranded siRNA conjugates for each group were serially diluted with PBS to 10 μM working solution (based on siRNA). 1 μL of siRNA conjugate working solution was added to each well of the 12-well culture plate, equivalent to a final transfection concentration of 10 nM (based on siRNA). Two wells were used for each siRNA concentration. Two additional wells were used as blank control wells with 1 μL / well of PBS added. RNA extraction, reverse transcription, and quantitative real-time PCR were performed as described above. Gene expression differences were calculated using the ΔΔCt method.

[0530] Table 10 Primer sequence information in Example 4

[0531] The results of Example 4 show that, compared with the reference conjugate RZ597002, the conjugate RZ597101 containing (NM130) in the positive chain has comparable or better inhibitory activity (Figure 4, Table 11).

[0532] Table 11. Inhibitory activity of siRNA conjugate on the target gene ANGPTL3 in primary mouse liver cells.

[0533] Example 5 evaluates the inhibitory activity of an antisense strand (NM130)-containing siRNA conjugate against the target gene CFB in mice.

[0534] This embodiment uses the "Method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice" to evaluate the inhibitory activity of the antisense strand conjugates RZm11504 and RZm11507 containing (NM130) and the reference conjugates RZm11001 and RZm11502 without (NM130) on the target gene CFB in mice.

[0535] Compared to the reference conjugate RZm11001, the antisense strand in conjugate RZm11504 is replaced with (NM130) at the 14th nucleotide starting from the 5' end.

[0536] Compared to the reference conjugate RZm11502, the antisense strand in conjugate RZm11507 is replaced with (NM130) at the 14th nucleotide starting from the 5' end.

[0537] Six- to eight-week-old C57BL / 6j mice were randomly divided into five groups of 15 mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the siRNA conjugate experimental group, the dosage was 3 mg (siRNA) / kg (mice), with a dosage volume of 5 mL / kg (mice). In the PBS control group, the dosage volume was 5 mL / kg (mice). The day of administration was designated D0. Five mice from each group were sacrificed on D7, D28, and D56. Gross dissections were performed, and liver tissue was collected and cut into several 2 mm segments. 3 Small pieces are preserved using RNAlater.

[0538] RNA extraction, reverse transcription, and real-time PCR detection were performed as described above. Gene expression differences were calculated using the ΔΔCt method. Primers are shown in Example 1.

[0539] The results of Example 5 show that, compared with the reference conjugates RZm11001 and RZm11502, the antisense conjugates RZm11504 and RZm11507 containing (NM130) have a longer duration of action and significantly higher inhibitory activity at D28 and D56 compared with the reference (Figure 5, Table 12).

[0540] Table 12 Inhibitory activity of siRNA conjugate against target gene CFB in mice.

[0541] Example 6 evaluates the inhibitory activity of an antisense strand containing (NM130) siRNA conjugate against the target gene complement C4B (C4B) in mice.

[0542] This embodiment uses the "Method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice" to evaluate the inhibitory activity of the antisense conjugate RZm12503 containing (NM130) and the reference conjugate RZm12501 without (NM130) on the target gene C4B in mice.

[0543] Compared to the reference conjugate RZm12501, the antisense strand in conjugate RZm12503 is replaced with (NM130) at the 9th nucleotide position starting from the 5' end.

[0544] Six- to eight-week-old C57BL / 6j mice were randomly divided into three groups of 15 mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the siRNA conjugate experimental group, the dosage was 3 mg (siRNA) / kg (mice), with a dosage volume of 5 mL / kg (mice). In the PBS control group, the dosage volume was 5 mL / kg (mice). The day of administration was designated D0. Five mice from each group were sacrificed on D7, D28, and D56. The animals were grossly dissected, and liver tissue was collected and cut into several 2 mm segments. 3 Small pieces are preserved using RNAlater.

[0545] RNA extraction, reverse transcription reaction, and Real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method.

[0546] Table 13 Primer sequence information in Example 6

[0547] The results of Example 6 showed that, compared with the reference conjugate RZm12501, the antisense chain containing (NM130) conjugate RZm12503 had a longer duration of action and significantly higher inhibitory activity at D56 compared with the reference (Figure 6, Table 14).

[0548] Table 14. Inhibitory activity of siRNA conjugate against target gene C4B in mice.

[0549] Example 7 evaluates the inhibitory activity of a siRNA conjugate containing (NM130) in the sense strand against the target gene CFB in mice.

[0550] This embodiment uses the "Method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice" to evaluate the inhibitory activity of the positive strand conjugate RZm11510 containing (NM130) and the reference conjugates RZm11015 and RZm11509 without (NM130) on the target gene CFB in mice.

[0551] Compared to the reference conjugate RZm11015, in the reference conjugate RZm11509, the 15th nucleotide of the antisense strand starting from the 5' end is replaced by T(moe), and in the conjugate RZm11510, the 7th nucleotide of the sense strand starting from the 5' end is replaced by (NM130).

[0552] Six- to eight-week-old C57BL / 6j mice were randomly divided into four groups of 15 mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the siRNA conjugate experimental group, the dosage was 3 mg (siRNA) / kg (mice), with a dosage volume of 5 mL / kg (mice). In the PBS control group, the dosage volume was 5 mL / kg (mice). The day of administration was designated D0. Five mice from each group were sacrificed on D7, D28, and D56. The animals were grossly dissected, and liver tissue was collected and cut into several 2 mm segments. 3 The small fragments were preserved using RNAlater. RNA extraction, reverse transcription, and real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method. Primers were as shown in Example 1.

[0553] The results of Example 7 show that the conjugate RZm11510 containing the NM130 group has comparable inhibitory activity at D7, D28 and D56 compared with the reference conjugates RZm11015 and RZm11509 (Figure 7, Table 15).

[0554] Table 15. Inhibitory activity of siRNA conjugate on target gene CFB in mice.

[0555] Example 8 evaluates the inhibitory activity of a siRNA conjugate containing (NM130) in the sense strand against the target gene C4B in mice.

[0556] This embodiment uses the "Method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice" to evaluate the inhibitory activity of the positive strand conjugates RZm12505 and RZm12506 containing (NM130) and the reference conjugate RZm12504 without (NM130) on the target gene C4B in mice.

[0557] Compared to the reference conjugate RZm12504, in conjugate RZm12505, the 7th nucleotide starting from the 5' end is replaced with (NM130) in the positive strand, and in conjugate RZm12506, the 8th nucleotide starting from the 5' end is replaced with (NM130) in the positive strand.

[0558] Six- to eight-week-old C57BL / 6j mice were randomly divided into four groups of 15 mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. The siRNA conjugate experimental group received 3 mg (siRNA) / kg (mice) at a dose of 5 mL / kg (mice). The PBS control group received 5 mL / kg (mice). The day of administration was designated D0. Five mice from each group were sacrificed on D7, D28, and D56. The animals were grossly dissected, and liver tissue was collected and cut into several 2 mm sections. 3 The small fragments were preserved using RNAlater. RNA extraction, reverse transcription, and real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method. Primers are shown in Example 6.

[0559] The results of Example 8 show that the conjugates RZm12505 and RZm12506 have comparable inhibitory activity to the reference conjugate RZm12504 at D7, D28 and D56 (Figure 8, Table 16).

[0560] Table 16. Inhibitory activity of siRNA conjugate against target gene C4B in mice.

[0561] Example 9 evaluates the inhibitory activity of siRNA conjugates containing (NM054) at the 3' end of the antisense strand against the target gene SOD1 in primary mouse liver cells.

[0562] Example 9 used the "Method for evaluating the inhibitory activity of siRNA conjugates against target genes in primary mouse liver cells" to evaluate the inhibitory activity of conjugate RZ599034, which has (NM054) at the second nucleotide position (starting from the 3' end of the antisense strand), and reference conjugate RZ599001, which does not contain (NM054), against the target gene SOD1 in primary mouse hepatocytes.

[0563] Primary mouse hepatocytes with a cell mass of 2 × 10⁻⁶ 5 Cells / well were seeded into 12-well culture plates. Each group of siRNA conjugates was serially diluted with PBS to 10 μM and 1 μM working solutions (based on siRNA). 1 μL / well of each concentration of siRNA conjugate working solution was added to each well of the 12-well culture plate, equivalent to a final transfection concentration of 10 nM and 1 nM (based on siRNA). Three wells were set up for each concentration of siRNA conjugate. Three additional wells were prepared as blank control wells with 1 μL / well of PBS solution added. RNA extraction, reverse transcription, and Real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method. Primers were as shown in Example 3.

[0564] The results of Example 9 show that the sequence RZ599034 modified by NM054 at position 20 of the antisense strand has a stronger target gene repression effect than the reference sequence RZ599001 (Figure 9, Table 17).

[0565] Table 17 Inhibitory activity of siRNA conjugate on target gene SOD1 in primary mouse liver cells

[0566] Example 10 evaluates the inhibitory activity of siRNA conjugates containing (NM054) at the 3' end of the antisense strand against the target gene SOD1 in mice.

[0567] This embodiment uses the "Method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice" to evaluate the inhibitory activity of conjugate RZ599060, which has (NM054) at the second nucleotide position starting from the 3' end of the antisense strand, conjugate RZ599061, which has (NM054) at both the first and second nucleotide positions starting from the 3' end of the antisense strand, and reference conjugate RZ599001, which does not contain (NM054), on the target gene SOD1 in mice.

[0568] Six- to eight-week-old C57BL / 6j mice were randomly divided into four groups of 15 mice each, based on body weight. Each group received the aforementioned siRNA conjugate via subcutaneous abdominal administration. In the siRNA conjugate experimental group, the dosage was 3 mg (siRNA) / kg (mice), with a dosage volume of 5 mL / kg (mice). In the PBS control group, the dosage volume was 5 mL / kg (mice). The day of administration was designated D0. Five mice from each group were sacrificed on D7, D28, and D49. The animals were grossly dissected, and liver tissue was collected and cut into several 2 mm sections. 3 The small fragments were preserved using RNAlater. RNA extraction, reverse transcription, and quantitative real-time PCR were performed as described above, and gene expression differences were calculated using the ΔΔCt method. Primers are shown in Example 3.

[0569] The results of Example 10 show that, compared with the reference conjugate RZ599001 which does not contain (NM054), the conjugates RZ599060 and RZ599061 which contain (NM054) at the 3' end of the antisense chain have higher inhibitory activity at D7, D28 and D49, especially at D49 where the inhibitory activity is significantly higher (Figure 10, Table 18).

[0570] Table 18. Inhibitory activity against the target gene SOD1 in mice after administration of siRNA conjugates.

[0571] Example 11 evaluates the inhibitory activity of siRNA conjugates containing (NM054) at the 3' end of the antisense strand against the target gene ANGPTL3 in mice.

[0572] This embodiment uses the "Method for evaluating the inhibitory activity of siRNA conjugates on target genes in mice" to evaluate the inhibitory activity of the siRNA conjugate RZ597115 (with NM054 at the second nucleotide position starting from the 3' end of the antisense strand), the siRNA conjugate RZ597116 (with NM054 at both the first and second nucleotide positions starting from the 3' end of the antisense strand), and the reference siRNA conjugate RZ597114 (without NM054) on the target gene ANGPTL3 in mice.

[0573] Six- to eight-week-old C57BL / 6j mice were randomly divided into four groups of 15 mice each, based on body weight. Each group received an siRNA conjugate via subcutaneous abdominal administration. The siRNA conjugate experimental group received 3 mg (siRNA) / kg (mice) at a dose of 5 mL / kg (mice). The PBS control group received 5 mL / kg (mice). The day of administration was designated D0. Five mice from each group were sacrificed on D7, D28, and D56. The animals were grossly dissected, and liver tissue was collected and cut into several 2 mm segments. 3 The small fragments were preserved using RNAlater. RNA extraction, reverse transcription, and real-time PCR detection were performed as described above, and gene expression differences were calculated using the ΔΔCt method. Primers are shown in Example 4.

[0574] The results of Example 11 showed that, compared with the reference siRNA conjugate RZ597114 which does not contain (NM054), the siRNA conjugates RZ597115 and RZ597116 which contain (NM054) at the 3' end of the antisense strand had higher inhibitory activity at D7, D28 and D56 (Figure 11, Table 19).

[0575] Table 19 Inhibitory activity of siRNA conjugate on target gene ANGPTL3 in mice.

[0576] Example 12 evaluates the inhibitory activity of siRNA conjugates containing (NM054) at the 3' end of the antisense strand against SEAP protein in the serum of SEAP reporter gene tool mice.

[0577] In this embodiment, SEAP reporter gene tool mice were used to evaluate the effects of conjugate R303079, in which the first and second nucleotides of the antisense strand starting from the 3' end are (NM054), and reference conjugate RZ003087, which does not contain (NM054), on the relative expression of SEAP in the serum of SEAP reporter gene tool mice.

[0578] Construction of SEAP reporter gene tool mice: The AGT transcript sequence was inserted into a transposon plasmid (Shanghai Langjing Biotechnology Co., Ltd.). 1.6 mL of plasmid mixture (25 μg of transposon plasmid fused with AGT transcript and 25 μg of Super PiggyBac Transposase plasmid) was injected into mice via tail vein hydrodynamics within 3–5 s. A mouse model stably expressing the SEAP reporter gene was obtained after 2 weeks.

[0579] Female BALB / c-SEAP stable mice were divided into groups of five according to their SEAP levels. Each test group received a predetermined dose of the drug, supplemented by a saline control group. RZ003087 and R303079 sequences were administered via subcutaneous abdominal injection. The drug dosage was calculated based on body weight, with an administration volume of 10 mL / kg (mice body weight) and a dosage of 3 mg / kg (calculated as siRNA / mice body weight). The saline control group received 10 mL / kg (mice body weight) of saline solution without the siRNA conjugate. Serum samples were collected from mice in the saline and RZ003087 / R303079 administration groups before administration and at D7, D14, D21, D28, and D35 after administration. Phospha-Light was used to analyze the results. TM The SEAP reporter gene assay system (Thermo, T1017) was used to detect SEAP levels in the serum of mice in all groups.

[0580] The results of Example 13 showed that, compared with the reference conjugate RZ003087, the R303079 conjugate containing (NM054) at the 3' end of the antisense strand had a higher inhibitory effect on the expression of SEAP protein in serum at all time points and showed a longer protein inhibition level (Figure 12, Table 20).

[0581] Table 20 shows the relative expression levels of SEAP in the serum of SEAP reporter gene tool mice after siRNA conjugation.

[0582] Example 13 Evaluation of the inhibitory activity of different modified siRNA sequences on the target gene superoxide dismutase 1 (SOD1) in mouse primary hepatocytes

[0583] The inhibitory activity of the LNA-modified sequences RZ599206, RZ599207, cEt-modified sequence RZ599208, GNA-modified sequence RZ599209, and UNA-modified sequence RZ599210 on the target gene SOD1 was evaluated using mouse primary hepatocytes.

[0584] Primary hepatocyte isolation from mice: Primary hepatocytes were extracted from fresh liver tissue of C57BL / 6j mice. The specific procedure involved anesthetizing mice with an intraperitoneal injection of 10% chloral hydrate solution, fixing the mice, and disinfecting their abdomen and chest with 75% ethanol. Surgical instruments were sterilized, and the abdominal cavity was opened to expose the portal vein and inferior vena cava. A heparin cap was attached to an indwelling needle, which was then connected to a scalp vein needle attached to an infusion pump vial (0.5 mM EDTA HBSS perfusion solution). The needle was inserted through the inferior vena cava, and perfusion was performed at a rate of 120 drops / min. The portal vein was then cut open to allow the perfusion solution to flow out. Perfusion continued for 4 minutes, followed by 8 minutes of perfusion with 0.8 mg / mL type IV collagenase HBSS solution (Sigma, C5138) (containing 0.08% DNA I enzyme (Sigma, DN25)). The perfused liver was removed from the animal and treated with HBSS (containing Ca2+). + Mg2 + The liver was cleaned with MACGENE (CC016) and placed in a sterile culture dish. DMEM complete medium (DMEM medium + 10% serum) was added and the liver was shredded. The cell suspension was filtered through a cell sieve to remove undigested tissue and connective tissue. The suspension was centrifuged at 800 rpm for 3 min and the supernatant was discarded. DMEM complete medium was added again and the suspension was centrifuged again to obtain primary mouse hepatocytes.

[0585] Cell culture and transfection: Add DMEM complete medium to adjust cell density to 2×10^ 5 Primary mouse hepatocyte suspension was obtained by measuring cells / mL. The cells were then seeded into 12-well culture plates pre-coated with type I rat tail collagen (coating method according to SolarBio (C8062) instructions at 2 μg / mL). 2 (Concentration coating), the volume of cell suspension added was 1000 μL / well, i.e., the cell quantity was 2 × 10^ 5 Cells / wells. Dilute each group of siRNA with PBS to 5 μM working solution (based on siRNA). Add 1 μL / well of siRNA working solution to each of the above 12-well culture plates. This is equivalent to a final siRNA transfection concentration of 5 nM (based on siRNA) in each well, with 2 culture wells per group. Shake the culture plate to mix thoroughly. Incubate the culture plate in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0586] mRNA expression level detection:

[0587] RNA extraction: Add 1 mL of TRIZOL reagent to each well and pipette to lyse the cells, incubating at room temperature for 3 minutes. Then, add 200 μL of chloroform to each 1 mL TRIZOL sample, mix by hand-tilting the centrifuge tube, incubate at room temperature for 3 minutes, and then centrifuge at 4°C and 12000 rpm for 10 minutes. After separating the aqueous phase, transfer the supernatant (approximately 400 μL) to a centrifuge tube containing an equal volume of isopropanol, mix, and incubate at room temperature for 10 minutes. Centrifuge again at 4°C and 12000 rpm for 10 minutes to precipitate RNA. Discard the supernatant, wash the precipitate with 1 mL of RNase-free 75% ethanol, and then centrifuge at 4°C and 12000 rpm for 5 minutes to remove as much supernatant as possible. Dry the precipitate (air dry at room temperature or vacuum dry for 5-10 minutes), dissolve the RNA precipitate in an appropriate amount of RNase-free water, and store below -70°C.

[0588] Reverse transcription: Take 1 μg of total RNA and use a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) with Oligo(dT)18 reverse transcription primers. Prepare a 20 μL reverse transcription system according to the instructions of the reverse transcription kit 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.

[0589] Real-time quantitative PCR: Next, a real-time quantitative PCR kit (ABI, SYBR) was used. TM The expression level of the target gene SOD1 mRNA in hepatocytes was detected using Select Master Mix (Catalog number: 4472908). 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. A 20 μL Real-time PCR reaction system was prepared for each PCR detection well according to the instructions of the real-time quantitative PCR kit. Each reaction system contained 5 μL of cDNA solution obtained from the above reverse transcription reaction and 10 μL of SYBR Green. TM 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 mixture in a real-time quantitative PCR instrument (ABI, StepOnePlus). TMReal-time PCR amplification was performed using a three-step method. The amplification program was 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. The denaturation, annealing, and extension process was repeated for 40 cycles.

[0590] The relative quantification of target gene mRNA in each test group was performed using the ΔΔCt method. The calculation method is summarized below:

[0591] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0592] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0593] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0594] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0595] The relative residual expression level of the target gene mRNA in the test group = 2 - ΔΔCt(test group) × 100%

[0596] Using the control group as a baseline, the mRNA expression level of the target gene in the test group was normalized, and the residual mRNA expression level of the target gene in the control group was defined as 100%.

[0597] The inhibition rate of target gene mRNA in the test group = 100% - the relative expression level of target gene mRNA in the test group

[0598] All activity experimental data are based on It is noted that all experimental data were plotted and analyzed using GraphPad Prism 8.0 software.

[0599] The primer sequences are shown in Table 8.

[0600] The results of Example 13 show that sequences RZ599206 and RZ599207 containing LNA modification, sequence RZ599208 containing cEt modification, sequence RZ599209 containing GNA modification, and sequence RZ599210 containing UNA can achieve high levels of inhibition of target genes at a concentration of 5 nM, with inhibitory activity of 80% or higher (see Figure 13, Table 21).

[0601] Table 21 shows the inhibitory activity level of the target gene SOD1 in mouse primary hepatocytes after administration of the siRNA conjugate described in this embodiment.

[0602] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure.

Claims

1. A double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, characterized in that, The double-stranded oligonucleotide comprises a sense strand and an antisense strand, each strand having 16 to 30 nucleotides, and the sense strand and antisense strand are at least partially anticomplementary to form a double-stranded region having 16 to 21 base pairs; the double-stranded oligonucleotide has at least one dangling end, and the dangling end is not located at the 5' end of the antisense strand. The double-stranded region contains at least one [2'-R1-2'-R2] modified nucleotide; R1 represents the substituent group of the 2'-OH ribose ring in the substituted nucleotide, and R2 represents the substituent group of the 2'-H ribose ring in the substituted nucleotide; R1 and R2 are each independently selected from halogens, optionally substituted C1-C6 alkyl groups, or optionally substituted C1-C6 alkoxy groups.

2. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, In the double-stranded region, at least one nucleotide from the antisense strand at positions 2, 6, 9, 12, 14, and 16 (starting from the 5' end) and from the sense strand at positions 10-13 and 15 (starting from the 5' end) that are complementary to the antisense strand through base pairing is selected from nucleotides modified with [2'-R1-2'-R2]. Optionally, the second nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]. Optionally, the 6th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]. Optionally, the 9th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]. Optionally, the 14th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]. Optionally, the 16th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]. Optionally, the 14th and 16th nucleotides of the antisense strand, starting from the 5' end, are selected from nucleotides modified with [2'-R1-2'-R2]. Optionally, the nucleotide in the sense strand that pairs with the 10th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2]. Optionally, the nucleotide in the sense strand that pairs with the 11th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2]. Optionally, the nucleotide in the sense strand that pairs with the 12th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2]. Optionally, the nucleotide in the sense strand that pairs with the 13th nucleotide of the antisense strand starting from the 5' end through base complementarity is selected from nucleotides modified with [2'-R1-2'-R2]. Optionally, the nucleotide in the sense strand that is base-complementarily paired with the 15th nucleotide of the antisense strand starting from the 5' end is selected from nucleotides modified with [2'-R1-2'-R2].

3. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, At least five nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, are each independently selected from nucleotides modified with [2'-R1-2'-R2] or nucleotides modified with 2'-fluorine, and the antisense strand contains no more than five nucleotides modified with 2'-fluorine; and / or, at least four nucleotides at positions 10-13 and 15 of the sense strand, which are base-complementary to the antisense strand, are each independently selected from nucleotides modified with [2'-R1-2'-R2] or nucleotides modified with 2'-fluorine, and the sense strand contains no more than four nucleotides modified with 2'-fluorine. Furthermore, among the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand (starting from the 5' end) and the nucleotides at positions 10-13 and 15 of the sense strand (starting from the 5' end) that are base-complementary, at least one nucleotide is selected from a nucleotide modified with [2'-R1-2'-R2].

4. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, characterized in that, The second nucleotide starting from the 5' end of the antisense strand is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four nucleotides from the 6th, 9th, 12th, 14th, and 16th positions are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five. Optionally, the 6th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2]; at least four of the nucleotides at positions 2, 9, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five. Optionally, the 14th nucleotide starting from the 5' end of the antisense strand is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 9, 12, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five. Optionally, the 16th nucleotide of the antisense strand, starting from the 5' end, is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 9, 12, and 14 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five. Optionally, the 14th and 16th nucleotides of the antisense strand, starting from the 5' end, are selected from nucleotides modified with [2'-R1-2'-R2], and the nucleotides at positions 2, 6, 9, and 12 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five. Optionally, the 9th nucleotide starting from the 5' end of the antisense strand is selected from a nucleotide modified with [2'-R1-2'-R2], and at least four of the nucleotides at positions 2, 6, 12, 14, and 16 are selected from nucleotides modified with 2'-fluorine; and the number of nucleotides modified with 2'-fluorine in the antisense strand does not exceed five.

5. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that, The nucleotide in the sense strand that pairs with the 10th nucleotide starting from the 5' end of the antisense strand via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2]. At least three of the nucleotides in the sense strand that pair with the 11th-13th and 15th nucleotides starting from the 5' end of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine. And the sense strand contains no more than four nucleotides modified with 2'-fluorine. Optionally, the nucleotide in the sense strand that pairs with the 11th nucleotide of the antisense strand starting from the 5' end via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th, 12th-13th, and 15th nucleotides of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine. Optionally, the nucleotide in the sense strand that pairs with the 12th nucleotide of the antisense strand starting from the 5' end via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th-11th, 13th, and 15th nucleotides of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine. Optionally, the nucleotide in the sense strand that pairs with the 13th nucleotide starting from the 5' end of the antisense strand via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th-12th and 15th nucleotides starting from the 5' end of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine. Optionally, the nucleotide in the sense strand that pairs with the 15th nucleotide starting from the 5' end of the antisense strand via base complementarity is selected from nucleotides modified with [2'-R1-2'-R2], and at least three of the nucleotides in the sense strand that pair with the 10th-13th nucleotides starting from the 5' end of the antisense strand via base complementarity are selected from nucleotides modified with 2'-fluorine; and the sense strand contains no more than four nucleotides modified with 2'-fluorine.

6. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, characterized in that, R1 and R2 are each independently selected from halogens or optionally substituted C1-C6 alkoxy groups; Optionally, R1 is selected from F or methoxy, and R2 is selected from F or methyl; Optionally, the [2'-R1-2'-R2] modified nucleotide is selected from the [2'-F-2'-F] modified nucleotide, the [2'-F-2'-CH3] modified nucleotide, or the [2'-OCH3-2'-CH3] modified nucleotide.

7. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The double-stranded oligonucleotide has at least one overhang, the overhang being composed of 1-3 modified nucleotides, and at least one of the overhangs being a [2'-R1-2'-R2] modified nucleotide; Optionally, the double-stranded oligonucleotide has two dangling ends, and the dangling ends are located at the 3' end of the antisense strand and the 3' end of the sense strand, respectively. Optionally, the double-stranded oligonucleotide has a dangling end, and the dangling end is located at the 3' end of the antisense strand; Optionally, the double-stranded oligonucleotide has a dangling end, and the dangling end is located at the 3' end of the positive strand; Optionally, the overhang is composed of 1-3 [2'-R1-2'-R2] modified nucleotides; Optionally, the overhang is composed of two [2'-R1-2'-R2] modified nucleotides; Optionally, the overhang is composed of two [2'-F-2'-CH3] modified nucleotides.

8. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-7, characterized in that, The double-stranded oligonucleotide contains at least three different modified nucleotides; preferably, the double-stranded oligonucleotide contains at least four different modified nucleotides; wherein the double-stranded oligonucleotide includes a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, and a fourth modified nucleotide; The first modified nucleotide is selected from nucleotides modified with [2'-R1-2'-R2]. The second modified nucleotide is selected from 2'-O-(CH2). n -R3 modified nucleotide; n is selected from integers from 0 to 3, and R3 is selected from optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxy, or... R 3a R 3b and R 3c Each is independently selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; The third modified nucleotide includes at least one of a 2'-F modified nucleotide or a 2'-deoxy modified nucleotide; The fourth modified nucleotide includes Acyclic modified nucleotides, At least one of the following; wherein the acyclic modified nucleotides include At least one of them; Where Base represents a nucleoside base, and Z is independently selected from -OH or -SH; R 4a and R 4b Each is independently selected from H or optionally substituted C1-C3 alkyl groups; j1 is selected from 1, 2 or 3; R 5a and R 5b Each is independently selected from H or optionally substituted C1-C3 alkyl groups; j2 is selected from 1, 2 or 3; j3 is selected from 1, 2, 3 or 4; j4 is selected from 1, 2, 3 or 4; R6 is selected from H or optionally substituted C1-C3 alkyl groups; R7 is selected from H, optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl or optionally substituted C10-C22 alkynyl; R8 is selected from Optionally substituted C10-C22 alkyl, optionally substituted C10-C22 alkenyl, or optionally substituted C10-C22 alkynyl; Optionally, the first modified nucleotide includes at least one of the following: a nucleotide modified with [2'-F-2'-F], a nucleotide modified with [2'-F-2'-CH3], or a nucleotide modified with [2'-OCH3-2'-CH3]. Optionally, the second modified nucleotide includes at least one of the following: a nucleotide modified with 2'-O-CH3, a nucleotide modified with 2'-O-CH2-O-CH2-CH3, a nucleotide modified with 2'-O-CH2-O-CH2-CF3, a nucleotide modified with 2'-O-CH2-CH2-O-CH3(2'-O-MOE), a nucleotide modified with 2'-O-TBDMS, a nucleotide modified with 2'-O-TIPS, or a nucleotide modified with 2'-O-TOM. Optionally, the fourth modified nucleotide includes RNA. LNA CeNA GNA A or HNA At least one of them; Optionally, the fourth modified nucleotide includes at least one of LNA, CeNA, GNA, or UNA.

9. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, characterized in that, The double-stranded oligonucleotide further includes 5'-terminal phosphorylation modification, which includes 5'-(E)-vinylphosphonate (5'-(E)-VP) modification, 5'-methylphosphonate (5'-MP) modification, (S)-5'-C-methyl analog modification, and 5'-thiophosphate (5'-PS) modification or analogues thereof.

10. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8 or 9, characterized in that, The antisense strand comprises at least three of a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, or a fourth modified nucleotide; Optionally, each nucleotide of the antisense strand is independently selected from [2'-F-2'-F] modified nucleotides, [2'-F-2'-Me] modified nucleotides, [2'-OMe-2'-Me] modified nucleotides, 2'-O-CH3 modified nucleotides, 2'-O-MOE modified nucleotides, 2'-F modified nucleotides, 2'-deoxy modified nucleotides, LNA, CeNA, or RNA; Optionally, when the modification of the nucleotide on the antisense strand includes at least one of [2'-F-2'-F] modification, 2'-deoxy modification or RNA, it is located at at least one of the 2nd, 4th, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand starting from the 5' end; Optionally, when the modification of the nucleotide on the antisense strand includes at least one of [2'-F-2'-Me] modification or [2'-OMe-2'-Me] modification, it is located at at least one of the 1st to 3rd positions of the antisense strand starting from the 3' end; Optionally, when the modification of the nucleotide on the antisense strand includes at least one of 2'-O-MOE modification, LNA or CeNA, it is located at one or more positions of the 5th-10th, 15th, 17th-19th positions of the antisense strand starting from the 5' end; Optionally, the modification of the nucleotide on the antisense strand is selected from at least 10 positions of the antisense strand, starting from the 5' end, when the 2'-O-methyl modification is performed; Optionally, when the nucleotide modification on the antisense strand is selected from 2'-F modification, it is located at at least four of the 2nd, 6th, 9th, 12th, 14th, and 16th positions of the antisense strand starting from the 5' end.

11. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 8-10, characterized in that, The antisense chain contains one or more of the following characteristics: 1) The first nucleotide of the antisense chain, starting from the 5' end, has at least one selected from 5'-(E)-vinylphosphonate (5'-(E)-VP) modification, 5'-methylphosphonate (5'-MP) modification, (S)-5'-C-methyl analog, and 5'-thiophosphate (5'-PS) modification or analogs thereof; preferably, the first nucleotide of the antisense chain, starting from the 5' end, has 5'-(E)-vinylphosphonate modification; more preferably, the first nucleotide of the antisense chain, starting from the 5' end, also has 2'-O-methyl modification; 2) The nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, include at least one of a [2'-F-2'-F] modified nucleotide, a 2'-deoxy modified nucleotide, or RNA, and the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, contain at least three 2'-F modified nucleotides; preferably, the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand, starting from the 5' end, include The antisense strand contains at least one of the [2'-F-2'-F] modified nucleotides or 2'-deoxy modified nucleotides, and the nucleotides at positions 2, 6, 9, 12, 14, and 16, starting from the 5' end, contain at least three 2'-F modified nucleotides, while the remaining nucleotides at positions 2, 6, 9, 12, 14, and 16 are 2'-O-methyl modified nucleotides; more preferably, the nucleotide at position 14, starting from the 5' end, is a [2'-F-2'-F] modified nucleotide; 3) The nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of a first modified nucleotide, a second modified nucleotide, or a fourth modified nucleotide; preferably, the nucleotides at positions 3-5, 7-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of a first modified nucleotide, a second modified nucleotide, or a fourth modified nucleotide. Or at least one of the acyclic modified nucleotides; more preferably, the nucleotides at positions 3-5, 7-10, 15, and 17-19, starting from the 5' end of the antisense strand, include at least one of the following: 2'-O-MOE modified nucleotide, 2'-O-TBDMS modified nucleotide, [2'-F-2'-Me] modified nucleotide, [2'-OMe-2'-Me] modified nucleotide, GNA, UNA, LNA, or cEt, and the antisense strand starting from the 5' end... The other nucleotides at positions 3-5, 7-10, 15, and 17-19, starting from the 5' end, are selected from nucleotides modified with 2'-O-methyl; more preferably, the nucleotides at positions 3-5, 7-10, 15, and 17-19, starting from the 5' end, of the antisense strand include a nucleotide modified with 2'-O-MOE, and the other nucleotides at positions 3-5, 7-10, 15, and 17-19, starting from the 5' end, of the antisense strand are selected from nucleotides modified with 2'-O-methyl. 4) At least one nucleotide at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, is selected from GNA or UNA; preferably, at least one nucleotide at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, is selected from GNA or UNA, and the other nucleotides at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3 modified nucleotides; more preferably, at least one nucleotide at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, is selected from GNA or UNA, and the other nucleotides at positions 5, 6, and 7 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3 modified nucleotides or 2'-F modified nucleotides; 5) The nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of a 2'-O-MOE modified nucleotide, a 2'-O-TBDMS modified nucleotide, a [2'-OMe-2'-Me] modified nucleotide, or an LNA, and the other nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3 modified nucleotides; preferably, the nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, include at least one of a 2'-O-MOE modified nucleotide, a 2'-O-TBDMS modified nucleotide, a [2'-OMe-2'-Me] modified nucleotide, or an LNA, and the other nucleotides at positions 5-10, 15, and 17-19 of the antisense strand, starting from the 5' end, are selected from 2'-O-CH3 modified nucleotides or 2'-F modified nucleotides; 6) The nucleotides at positions 11-13 of the antisense strand, starting from the 5' end, are each independently selected from at least one of a nucleotide modified with 2'-O-CH3 or a nucleotide modified with 2'-F; preferably, the nucleotides at positions 11-13 of the antisense strand, starting from the 5' end, are all nucleotides modified with 2'-O-CH3, or the nucleotides at positions 11 and 13 of the antisense strand, starting from the 5' end, are selected from nucleotides modified with 2'-O-CH3, and the nucleotide at position 12 is selected from nucleotides modified with 2'-F.

12. The double-stranded oligonucleotide according to any one of claims 8-11, characterized in that, Starting from the 5' end, the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from nucleotides modified with [2'-F-2'-F], nucleotides modified with 2'-F, or nucleotides modified with 2'-O-CH3, and contain at least three nucleotides modified with 2'-F. Preferably, the nucleotides at positions 2, 6, and 12 are nucleotides modified with 2'-F, and the nucleotides at positions 9, 14, or 16 are selected from nucleotides modified with [2'-F-2'-F], nucleotides modified with 2'-F, or nucleotides modified with 2'-O-CH3. The nucleotides at positions 1, 3-5, 7-8, 10-11, 13, and 15 of the antisense strand are selected from nucleotides modified with 2'-O-MOE or nucleotides modified with 2'-O-CH3; preferably, the nucleotide at position 15 is a nucleotide modified with 2'-O-MOE, and the nucleotides at positions 1, 3-5, 7-8, 10-11, and 13 are all nucleotides modified with 2'-O-CH3. The nucleotides at positions 17-19 or 17-21 are selected from nucleotides modified with 2'-O-MOE, [2'-OMe-2'-Me], [2'-F-2'-Me], or nucleotides modified with 2'-O-CH3; preferably, the nucleotide at position 17 is a nucleotide modified with 2'-O-CH3, and the nucleotides at positions 18 and 19 are selected from nucleotides modified with [2'-F-2'-Me], [2'-OMe-2'-Me], or nucleotides modified with 2'-O-CH3; or preferably, the nucleotides at positions 17-19 are nucleotides modified with 2'-O-CH3, and the nucleotides at positions 20 and 21 are selected from nucleotides modified with [2'-F-2'-Me], [2'-OMe-2'-Me], or nucleotides modified with 2'-O-CH3. Optionally, starting from the 5' end, the antisense strand further comprises one or more of the following: at least one 2'-deoxy modified nucleotide at position 9, 14, or 16; at least one of GNA or UNA in positions 5-7; 5'-(E)-vinylphosphonate modified nucleotide at position 1; and at least one of 2'-O-TBDMS modified nucleotide, GNA, UNA, LNA, or cEt in positions 3-5, 7-11, 13-15, 17-19, or 17-21.

13. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 8-12, characterized in that, The positive strand contains at least three of a first modified nucleotide, a second modified nucleotide, a third modified nucleotide, or a fourth modified nucleotide; Optionally, the positive strand contains a nucleotide modified with 2'-O-CH3 and a nucleotide modified with 2'-F, and in addition to the nucleotide modified with 2'-O-CH3 and the nucleotide modified with 2'-F, the positive strand also contains at least one of the following: a nucleotide modified with 2'-O-MOE, a nucleotide modified with [2'-F-2'-F], a nucleotide modified with [2'-F-2'-Me], a nucleotide modified with [2'-OMe-2'-Me], LNA, CeNA, DNA, or RNA. Optionally, when the nucleotide on the sense strand contains at least one of a [2'-F-2'-F] modified nucleotide, a 2'-deoxy modified nucleotide, or RNA, it forms a base pair with any nucleotide at positions 10-13 and 15 of the antisense strand, starting from the 5' end, through complementary base pairing. Optionally, when the nucleotide on the positive strand contains a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me], it is located at the 3' end or the 5' end of the positive strand; Optionally, when the nucleotide on the sense strand contains at least one of a 2'-O-MOE modified nucleotide, LNA, or cEt, it forms a base pair with any nucleotide at positions 1-9 and 14-19 of the antisense strand, starting from the 5' end, through complementary base pairing. Optionally, when the nucleotides on the sense strand contain at least 10 nucleotides modified with 2'-O-methyl, they form base pairs with any nucleotides at positions 1-10 and 14-19 of the antisense strand, starting from the 5' end, through complementary base pairing. Optionally, when the nucleotides on the sense strand contain at least three 2'-F modified nucleotides, they form base pairs with any nucleotides at positions 10-13 and 15 of the antisense strand, starting from the 5' end, through complementary base pairing. Optionally, the positive chain also has a thiophosphate modification at one or more of the following positions: between the first and second nucleotides starting from the 5' end, between the second and third nucleotides starting from the 5' end, between the first and second nucleotides starting from the 3' end, and / or between the second and third nucleotides starting from the 3' end; preferably, the positive chain also has a thiophosphate modification between the first and second nucleotides starting from the 5' end and between the second and third nucleotides starting from the 5' end; or, the positive chain also has a thiophosphate modification between the first and second nucleotides starting from the 5' end, between the second and third nucleotides starting from the 5' end, and between the first and second nucleotides starting from the 3' end.

14. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 8-13, characterized in that, Starting from the 3' end, at least one of the nucleotides at positions 1 and 2 of the antisense strand is selected from a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me]. Preferably, starting from the 3' end, the first nucleotide of the antisense strand is selected from a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me], and the second nucleotide is a nucleotide modified with 2'-O-CH3; or, starting from the 3' end, the second nucleotide of the antisense strand is selected from a nucleotide modified with [2'-F-2'-Me] or a nucleotide modified with [2'-OMe-2'-Me], and the first nucleotide is a nucleotide modified with 2'-O-CH3. More preferably, starting from the 3' end, the first and second nucleotides of the antisense strand are both selected from nucleotides modified with [2'-F-2'-Me] or nucleotides modified with [2'-OMe-2'-Me]. Most preferably, starting from the 3' end, the first and second nucleotides of the antisense strand are both selected from nucleotides modified with [2'-F-2'-Me]; or, starting from the 3' end, the first and second nucleotides of the antisense strand are both selected from nucleotides modified with [2'-OMe-2'-Me]; or, starting from the 3' end, the first nucleotide of the antisense strand is selected from nucleotides modified with [2'-OMe-2'-Me] and the second nucleotide is selected from nucleotides modified with [2'-F-2'-Me]; or, starting from the 3' end, the first nucleotide of the antisense strand is selected from nucleotides modified with [2'-F-2'-Me] and the second nucleotide is selected from nucleotides modified with [2'-OMe-2'-Me].

15. The double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-14, characterized in that, The double-stranded oligonucleotide or its pharmaceutically acceptable salt is selected from siRNA.

16. A conjugate, characterized in that, The conjugate comprises the double-stranded oligonucleotide of any one of claims 1-15 or a pharmaceutically acceptable salt thereof, and one or more targeted delivery ligands; Optionally, the targeted delivery ligand is selected from liver-targeted delivery ligands, kidney-targeted delivery ligands, lung-targeted delivery ligands, muscle-targeted delivery ligands, fat-targeted delivery ligands, heart-targeted delivery ligands, or central nervous system (CNS)-targeted delivery ligands; Optionally, the liver-targeted delivery ligand is a GalNAc ligand.

17. A composition, characterized in that, The composition comprises a double-stranded oligonucleotide as described in any one of claims 1-15 or a pharmaceutically acceptable salt thereof and / or a conjugate as described in claim 16.

18. Any of the following uses in the preparation of medicaments for the prevention and / or treatment of diseases: (I) the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-15; and / or (II) the conjugate as described in claim 16; and / or (III) The composition as described in claim 17.

19. The use of any of the following in the preparation of a drug for reducing the expression or activity of a target gene: (I) the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-15; and / or (II) the conjugate as described in claim 16; and / or (III) The composition as described in claim 17.

20. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any of the following and pharmaceutically acceptable excipients: (I) the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-15; and / or (II) the conjugate as described in claim 16; and / or (III) The composition as described in claim 17.

21. A method for reducing the expression or activity of a target gene, characterized in that, The method includes contacting the cells with any of the following: (I) the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1-15; and / or (II) the conjugate as described in claim 16; and / or (III) The composition of claim 17; and / or (IV) The pharmaceutical composition as described in claim 20.

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