Double-stranded oligonucleotide, conjugate and composition, and use therefor

Abstract

Provided are a double-stranded oligonucleotide, a conjugate and a composition, as well as a use therefor, relating to the technical field of nucleic acid drugs. The double-stranded oligonucleotide comprises a sense strand and an antisense strand, 17-23 nucleotides of the sense strand and the antisense strand being at least partially reverse complementary, to form a duplex region; the duplex region contains at least five phosphorothioate nucleotide linkages. The double-stranded oligonucleotide has at least one overhang, and the overhang is not located at the 5' end of the antisense strand; the overhang comprises at least one nucleotide modified with [2'-R1-2'-R2].

Description

Double-stranded oligonucleotides, conjugates and compositions and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202411259745.X, filed on September 9, 2024, and entitled “Double-stranded oligonucleotides, conjugates and compositions and uses thereof”, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the technical field of nucleic acid drugs, and in particular to a double-stranded oligonucleotide, a conjugate and a conjugate composition containing the same, and uses thereof. BACKGROUND

[0004] The following statements are merely provided to relate to the background of the present disclosure, and do not necessarily constitute the prior art.

[0005] Double-stranded oligonucleotides are a class of drugs artificially synthesized by chemical methods, which act on mRNA through base complementary pairing, interfere with the unwinding, replication, transcription, splicing processing of mRNA, and even the output and translation of genes, so that the abnormal coding genes lose function, and then prevent the expression of “wrong” proteins, thereby playing a unique mechanism of regulating the transcription and translation process of disease genes at the gene level.

[0006] Due to the unique chemical structure of oligonucleotide drugs, they exhibit poor drugability: large molecular weight, strong hydrophilicity, high negative charge, do not follow Lipinski's principle, and also have poor pharmacokinetic characteristics, cannot pass through biological membranes, and have off-target effects.

[0007] Unmodified double-stranded oligonucleotides are negatively charged, highly polar, highly water-soluble (in neutral and alkaline environments), difficult to pass through lipid bilayers and blood-brain barriers and other biological membranes, have poor distribution characteristics, poor stability, are easily degraded in extracellular fluid, are easily cleared by the kidneys and reticuloendothelial system (such as mononuclear phagocytes, liver sinusoidal endothelial cells, and Kupffer cells), have poor affinity for target sites, are prone to off-target effects, and the like. Therefore, drugs with double-stranded oligonucleotides as active ingredients still face problems such as low transfection efficiency, off-target effects, and delivery obstacles. Therefore, it is necessary to improve drugability by means of chemical modification or drug delivery systems. Through chemical modification, the resistance of the drug to nucleases can be improved, the binding force with mRNA can be increased, and the toxic side effects can be reduced. Although chemical modification improves the stability of the drug, it simultaneously reduces the affinity of the drug to the target gene, and therefore optimizing the modification method of double-stranded oligonucleotides can improve their use effect.

[0008] In view of this, the present disclosure is proposed. SUMMARY

[0009] The present disclosure aims to provide double-stranded oligonucleotides, conjugates and compositions and their uses to improve the affinity of double-stranded oligonucleotides to target genes and the inhibition efficiency of target genes.

[0010] To solve the above technical problems, the present disclosure adopts the following technical solutions:

[0011] In a first aspect, the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand comprises 17-25 consecutive nucleotides in the mRNA expressed by a target gene, and 17-23 nucleotides in the sense strand and the antisense strand are at least partially reverse complementary to form a duplex region;

[0012] wherein at least 5 phosphorothioate internucleoside linkages exist in the duplex region;

[0013] At least one overhang exists in the double-stranded oligonucleotide, and the overhang is not located at the 5' end of the antisense strand; the overhang comprises at least one [2'-R1-2'-R2] modified nucleotide, R1 represents a substituent group replacing 2'-OH of the ribose ring in the substituted nucleotide, R2 represents a substituent group replacing 2'-H of the ribose ring in the substituted nucleotide, R1 is selected from halogen, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy, and R2 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy.

[0014] In a second aspect, the present disclosure provides a conjugate comprising the double-stranded oligonucleotide of the first aspect and one or more target delivery ligands capable of binding to cell surface receptors.

[0015] In a third aspect, the present disclosure provides a composition comprising the conjugate of the second aspect.

[0016] In a fourth aspect, the present disclosure provides the use of any of the following in the preparation of a medicament for preventing and / or treating a disease:

[0017] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0018] (II) the conjugate of the second aspect; and / or

[0019] (III) the composition of the third aspect.

[0020] In a fifth aspect, the present 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 of the first aspect; and / or

[0022] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0023] (III) the composition of the third aspect.

[0024] In a sixth aspect, the present disclosure provides a pharmaceutical composition comprising any of the following and a pharmaceutically acceptable excipient:

[0025] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0026] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0027] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0028] In a seventh aspect, the present disclosure provides a method of reducing the expression or activity of a target gene, the method comprising contacting a cell with any of the following:

[0029] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0030] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0031] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0032] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0033] The present disclosure has the following beneficial effects:

[0034] The present disclosure improves the affinity of the double-stranded oligonucleotide to the target gene and the inhibitory ability of the double-stranded oligonucleotide to the target gene, and can significantly reduce the activity of the target gene in the subject. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present disclosure, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0036] Figure 1 shows the inhibitory activity of the target gene in mouse liver primary cells after administration of siRNA conjugate RZ599034 and reference sequence, respectively, in Example 1;

[0037] Figure 2 shows the inhibitory activity of the target gene in mice after administration of siRNA conjugates RZ599060, RZ599061 and reference sequence, respectively, in Example 2;

[0038] Figure 3 shows the inhibition activity of target genes in mice after administration of siRNA conjugates RZ597115, RZ597116 and control conjugate, respectively, in Example 3;

[0039] Figure 4 shows the inhibition activity of target genes in mouse primary hepatocytes after administration of siRNA conjugates RZ502053-RZ502068 and reference sequence, respectively, in Example 4

[0040] Figure 5 shows the inhibition activity of target genes in mouse primary hepatocytes after administration of siRNA conjugates RZ502069-RZ502084 and reference sequence, respectively, in Example 4;

[0041] Figure 6 shows the inhibition activity of target genes in mice after administration of siRNA conjugate RZ011307 and reference sequence, respectively, in Example 5;

[0042] Figure 7 shows the relative expression level of SEAP in serum of single-dose mice after administration of siRNA conjugate RZ003075 and reference sequence, respectively, in Example 6;

[0043] Figure 8 shows the relative expression level of SEAP in serum of single-dose mice after administration of siRNA conjugate RZ003079 and reference sequence, respectively, in Example 7;

[0044] Figure 9 shows the relative expression level of SEAP in serum of single-dose mice after administration of siRNA conjugate RZ003080 and reference sequence, respectively, in Example 8;

[0045] Figure 10 shows the relative expression level of SEAP in serum of single-dose mice after administration of siRNA conjugate RZ003076 and reference sequence, respectively, in Example 9. DETAILED DESCRIPTION

[0046] The technical solutions of the present disclosure will be described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present disclosure.

[0047] Explanation of terms:

[0048] The following definitions shall apply unless otherwise indicated. For purposes of the present disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, and the "Handbook of Chemistry and Physics" 75thEd., 1994. Additionally, general principles of organic chemistry are described 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.

[0049] As used herein, a double-stranded oligonucleotide is composed of two strands, one of which is complementary to a target sequence and is referred to as the antisense or guide strand, and the other of which is referred to as the sense or passenger strand.

[0050] As used herein, "antisense strand" includes a region that is at least partially reverse complementary to a target sequence. "Sense strand" refers to a strand of RNA that contains a sequence that is substantially complementary to an antisense strand.

[0051] As used herein, "partially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved, for example, 3, 2, 1, or 0.

[0052] As used herein, "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand each pair with a base on the other strand in a complementary manner.

[0053] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a product of processing of the primary transcript. The target gene can be in a cell, e.g., a cell in a subject.

[0054] As used herein, the term "subject" refers to any animal which is to be examined, investigated, or treated, and is not intended to be limited to any particular type of subject. In some embodiments of the present disclosure, a human is the preferred subject, while in other embodiments a non-human animal is the preferred subject, including but not limited to mice, monkeys, ferrets, cows, sheep, goats, pigs, chickens, turkeys, dogs, cats, horses, and reptiles.

[0055] As used herein, "overhanging end" (also referred to as "sticky end") refers to at least one non-base-paired nucleotide that overhangs from a double helical structure (e.g., dsRNA). For example, an overhanging end can occur when the 3' end of one strand of a sense strand and / or an antisense strand overhangs the 5' end of the other strand, or when the 5' end of one strand of a sense strand and / or an antisense strand overhangs the 3' end of the other strand. The overhanging end can comprise at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The overhanging end can occur at the 5' end of the sense strand, and / or at the 3' end of the sense strand, and / or at the 3' end of the antisense strand.

[0056] As used herein, "phosphodiester internucleoside linkage" and "phosphodiester linkage" are used interchangeably and have the structure:

[0057] As used herein, "phosphorothioate internucleoside linkage" and "phosphorothioate linkage" are used interchangeably and have the structure:

[0058] As used herein, "comprising" or "including," or "having" means including but not limited to, and thus specifies the presence of stated elements, integers, or steps, but not to the exclusion of any other elements, integers or steps. It is further to be understood that where this term is used, unless specifically stated to the contrary, it is intended that the completing group of elements, integers or steps is presented and that step plus additional steps which are either recited more

[0059] As used herein, "optionally," "optional," "optional" or "may" means that the subsequently described event or circumstance can or can not occur, and thus the description includes instances where the event or circumstance occurs and instances where it does not.

[0060] As used herein, the term "optionally substituted" is used to define a variable that can be either unsubstituted or substituted.

[0061] As used herein, the term "unsubstituted" means that the specified group bears no substituents.

[0062] As used herein, the terms "substituted," "substituted," and "substitution" are used interchangeably and mean that any one or more hydrogen atoms in the given structure are replaced with a specified substituent (e.g., C 1-6 alkyl, C 1-6 alkoxy, or halogen) provided that the designated atom's normal valence is not exceeded, and that the substitution results in a stable compound. Unless otherwise specified, a substituted group can have a substituent at each substitutable position that the group can bear. When more than one position in the given structure can be substituted with one or more substituents selected from specified groups, then those selected substituents can be the same or different at each position of substitution.

[0063] Herein, the terms "each independently selected from" and "independently selected from" are interchangeable, and should be interpreted broadly, meaning that the specific options expressed by the same symbol among different groups do not affect each other, and the specific options expressed by the same symbol among the same group do not affect each other.

[0064] Herein, "halogen" or "halo" refers to any one of the radio-stable atoms of group 7 of the periodic table of elements, such as fluorine, chlorine, bromine, or iodine; wherein, fluorine and chlorine are preferred, and fluorine is more preferred.

[0065] Herein, "hydroxyl" refers to an -OH group.

[0066] Herein, "alkyl" refers to a straight or branched chain alkyl group that is completely saturated (i.e., no double or triple bonds). "C1-C6 alkyl" means that the alkyl group has from 1 to 6 carbon atoms in the alkyl chain (whenever a numerical range is given, the range is inclusive of the numbers given; e.g., "1 to 6 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms, but this definition also covers the term "alkyl" where no numerical range is designated). Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, and the like. The alkyl group can be substituted or unsubstituted.

[0067] Herein, "alkoxy" refers to the group -OR, where R is an alkyl group as defined above, such as "C1-C6 alkoxy", which includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and t-butoxy, and the like.

[0068] Herein, when a letter refers to an object or quantity, the case is distinguished, e.g., B and b represent different referring objects or quantities.

[0069] Herein, "2'-halogen-modified nucleotide" refers to a nucleotide in which the 2' position of the ribosyl group is substituted with a halogen atom. Exemplarily, a 2'-F-modified nucleotide.

[0070] Herein, "2'-deoxy-modified nucleotide" refers to a nucleotide in which the 2' position of the ribosyl group is substituted with a hydrogen atom.

[0071] Herein, "2'-O-R3-modified nucleotide" refers to a nucleotide in which the 2' position of the ribosyl group is substituted with 2'-O-R3.

[0072] Herein, "2'-O-(CH2)n "R4-modified nucleotide" refers to a nucleotide in which the ribosyl 2' position hydroxyl is replaced with a 2'-0-(CH2) n "R4-substituted nucleotide" refers to a nucleotide in which the ribosyl 2' position hydroxyl is replaced with a 2'-0-(CH2)

[0073] In the present disclosure, the structure of TBDMS is In the present disclosure, the structure of TIPS is In the present disclosure, the structure of TOM is

[0074] In the present disclosure, "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates the targeted cleavage of RNA transcripts of the RISC pathway by forming a silencing complex (RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through a known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and conversion into proteins.

[0075] In the present disclosure, "conjugate", "linked", and "conjugate linked" are used interchangeably, and refer to the covalent linkage between two or more chemical moieties, each having a specific function; correspondingly, "conjugate" refers to a compound formed by the covalent linkage between two or more chemical moieties. Further, "drug conjugate" refers to a compound formed by the covalent linkage of one or more chemical moieties having specific functions to an active drug. In the present disclosure, "drug conjugate" of the present disclosure is also referred to as "conjugate", "double-stranded oligonucleotide conjugate", or "siRNA conjugate" sometimes, especially in the examples. Drug conjugate should be understood as the general term of drug conjugate or a specific drug conjugate represented by a specific structural formula, depending on the context.

[0076] In the present disclosure, "targeted delivery ligand", "delivery ligand", "ligand", "targeted delivery carrier", "delivery carrier", and "carrier" are used interchangeably, and generally refer to any compound or molecule capable of covalently or otherwise chemically binding to a biologically active substance, such as an oligonucleotide. In certain embodiments, the ligand is capable of directly or indirectly interacting with another compound, for example a receptor, which can be present on the surface of a cell, or alternatively can be an intracellular and / or intercellular receptor, and the interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or binding.

[0077] As used herein, the "central nervous system" (CNS) is composed of the brain and spinal cord (the brain and spinal cord are the central parts of various reflex arcs).

[0078] As used herein, the terms "treat," "treating," or "treatment" can be used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. Here, a therapeutic benefit is achieved with regard to the underlying disorder by observing an improvement in one or more physiological symptoms associated with the underlying disorder, even though the subject can still be afflicted with the underlying disorder.

[0079] As used herein, the terms "prevent," "preventing," or "prevention" can be used interchangeably herein, and refer to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To achieve a "prophylactic benefit," a double-stranded oligonucleotide, conjugate, or composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the subject can not yet be diagnosed with the disease.

[0080] As used herein, the terms "reduce," "inhibit," "decrease," "silence," "down- regulate," "suppress," and other similar terms can be used interchangeably, and include inhibition at any level. Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dosing baseline level or a level determined from a subject, cell, or sample that has never been treated or treated with a control, such as a buffer control or inert agent control.

[0081] As used herein, the term "contacting" generally refers to bringing two or more different types of substances together in any order, in any manner, and for any length of time. Contacting can occur in vivo, ex vivo, or in vitro. In some embodiments, it can refer to bringing an RNAi agent or composition of the disclosure into direct contact with a cell or tissue. In other embodiments, the term refers to bringing an RNAi agent or composition of the disclosure into indirect contact with a cell or tissue.

[0082] As used herein, "subject" generally refers to a human or non-human animal (including mammals), such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domesticated animals (dogs and cats), farm animals (horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs) in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease. Human subjects include fetal, neonatal, infant, juvenile, and adult human subjects. Subjects include animal models of disease.

[0083] 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.

[0084] In this article, "pharmaceutical composition" can refer to a drug used for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients that constitute one or more adjunct components. Typically, the composition is prepared by uniformly and adequately combining the active siRNA with liquid excipients, finely pulverized solid excipients, or both.

[0085] 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 the mammals to which it is treated.

[0086] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients is also within the scope of this disclosure, except for those that are incompatible with the siRNA of this disclosure, such as any adverse biological effects they may produce or interactions that may occur in a harmful manner with any other component of the pharmaceutically acceptable composition.

[0087] In addition to any conventional excipients, the use of any siRNA incompatible with the present disclosure, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition in a harmful manner, is also within the scope of this disclosure.

[0088] As used herein, the term "treatment" refers to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of a disease or condition. As used herein, "treatment" covers any administration of a drug or siRNA to an individual to treat, cure, heal, alleviate, relieve, or prevent a disease in the individual, including but not limited to administration of a drug comprising an siRNA or siRNA conjugate described herein to an individual in need thereof.

[0089] As used herein, "effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to effect any one or more of the beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing risk, lessening severity, or delaying the onset of a condition, including biochemical, histological and / or behavioral symptoms of the condition, its complications and intermediate pathological phenotypes presenting during development of the condition. For therapeutic use, beneficial or desired results include clinical results such as decreasing the incidence of various disease states associated with the target gene, target mRNA, or target protein of the disclosure, or ameliorating one or more symptoms of the disease state, reducing the dose of other medications required to treat the disease state, enhancing effect of another medication, and / or delaying the progression of the disease state in a patient.

[0090] As used herein, "double-stranded oligonucleotide" and "conjugate" can be obtained by methods of preparation conventional in the art, such as methods of solid-phase synthesis and liquid-phase synthesis. Among them, solid-phase synthesis has been commercialized as a subscription service. Methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into double-stranded oligonucleotides of the disclosure can be known to those skilled in the art by using nucleoside monomers with corresponding modifications.

[0091] As used herein, the term "chemical modification" or "modification" includes all changes of a nucleic acid by chemical means, such as addition or removal of a chemical moiety, or substitution of one chemical moiety with another chemical moiety.

[0092] As used herein, "base" includes any known DNA and RNA bases, base analogs, such as purines or pyrimidines, which also include natural compounds adenine, thymine, guanine, cytosine, uracil, hypoxanthine, and natural analogs.

[0093] 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 some isomer forms for simplicity, this disclosure can include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0094] 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.

[0095] In this article, the term "chirality" refers to a molecule that has the property that it cannot be superimposed on its mirror image; while "achirality" refers to a molecule that can be superimposed on its mirror image.

[0096] In this article, the term "enantiomer" refers to two non-overlapping but mirror-image isomers of a compound.

[0097] 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.

[0098] Unless otherwise stated, the base composition and modifications in this disclosure have the following meanings: uppercase letters A, U, G, C, and T represent the base composition of nucleotides; lowercase letter d indicates that the nucleotide represented by the uppercase letter to its left is a 2'-deoxy modified nucleotide; lowercase letter m indicates that the nucleotide represented by the uppercase letter to its left is a 2'-O-methyl modified nucleotide; lowercase letter f indicates that the nucleotide represented by the uppercase letter to its left is a 2'-fluoro modified nucleotide; (moe) indicates that the nucleotide represented by the uppercase letter to its left is a 2'-O-methoxyethyl modified nucleotide; lowercase letter s indicates that the two nucleotides represented by the two adjacent letters to its left and right are linked by a phosphate thioester bond.

[0099] The structural formula of the 2'-deoxy-modified nucleotide is:

[0100] The structural formula of the nucleotide modified with 2'-O-methyl is:

[0101] 2'-fluoro-modified nucleotides have the structure:

[0102] 2'-O-methoxyethyl-modified nucleotides have the structure:

[0103] [2'-F-2'-F] modified nucleotides have the structure:

[0104] where Base represents a nucleobase A, U, G, C or T.

[0105] (NM054) represents a nucleotide having the structure:

[0106] (NM118) represents a nucleotide having the structure:

[0107] (NM130) represents a nucleotide having the structure:

[0108] (NM131) represents a nucleotide having the structure:

[0109] (CR01008x3) represents a targeted delivery ligand having the structure:

[0110] (CR01008x4) represents a targeted delivery ligand having the structure:

[0111] L96 represents a targeted delivery ligand having the structure:

[0112] A double-stranded oligonucleotide:

[0113] In a first aspect, there is provided a double-stranded oligonucleotide comprising a sense strand and an antisense strand, the nucleotide sequence of the sense strand comprising 17-25 consecutive nucleotides in an mRNA of a target gene expression, and 17-23 nucleotides in the sense strand and the antisense strand being at least partially reverse-complementary to form a duplex region.

[0114] wherein the duplex region has at least 5 phosphorothioate intemucleosidic linkages;

[0115] The double stranded oligonucleotide has at least one overhang, and the overhang is not located at the 5' end of the antisense strand; the overhang comprises at least one [2'-R1-2'-R2] modified nucleotide, R1 represents a substituent group replacing the 2'-OH of the ribose ring in a substituted nucleotide, R2 represents a substituent group replacing the 2'-H of the ribose ring in a substituted nucleotide, R1 is selected from halogen, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy, and R2 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy.

[0116] In alternative embodiments, the duplex region contains 5, or 6, or 7, or 8, or 9, or 10 phosphorothioate internucleosidic linkages.

[0117] In alternative embodiments, the duplex region contains at least 5 phosphorothioate internucleosidic linkages; wherein the antisense strand contains at least 3 phosphorothioate internucleosidic linkages in the duplex region, and the sense strand contains at least 2 phosphorothioate internucleosidic linkages in the duplex region.

[0118] In alternative embodiments, the antisense strand contains at least 3 phosphorothioate internucleosidic linkages in the duplex region in the 5' to 3' direction, and the 3 phosphorothioate internucleosidic linkages are located between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, and between the 10th and 11th nucleotides of the antisense strand, respectively.

[0119] In alternative embodiments, the sense strand contains at least 2 phosphorothioate internucleosidic linkages in the duplex region in the 5' to 3' direction, and the 2 phosphorothioate internucleosidic linkages are located between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides of the sense strand in the duplex region.

[0120] In alternative embodiments, the nucleotides of the sense strand that base pair with the 10th nucleotide of the antisense strand and the nucleotides that base pair with the 11th nucleotide of the antisense strand are linked by phosphodiester bonds.

[0121] In alternative embodiments, at least one of the internucleosidic linkages between the overhang and the duplex region, and the internucleosidic linkages between the nucleotides in the overhang of the double stranded oligonucleotide is a phosphorothioate linkage.

[0122] In optional embodiments, the internucleoside linkages between the overhang and duplex region in the double stranded oligonucleotide, and between nucleotides in the overhang are selected from phosphorothioate diester linkages. In optional embodiments, the overhang consists of 1-5 [2'-R1-2'-R2] modified nucleotides; for example 1, 2, 3, 4, or 5, etc.

[0123] In optional embodiments, the overhang consists of 2-5 [2'-R1-2'-R2] modified nucleotides, and the two adjacent [2'-R1-2'-R2] modified nucleotides are linked by a phosphorothioate diester linkage.

[0124] In optional embodiments, the overhang consists of 2-5 [2'-R1-2'-R2] modified nucleotides, and the two adjacent [2'-R1-2'-R2] modified nucleotides are linked by a phosphorothioate diester linkage.

[0125] In optional embodiments, there is one overhang in the double stranded oligonucleotide, and the overhang is located at the 3' end of the antisense strand.

[0126] In optional embodiments, R1 is selected from halogen or optionally substituted C1-C6 alkoxy. In optional embodiments, R1 is selected from F or methoxy.

[0127] In optional embodiments, R2 is selected from optionally substituted C1-C6 alkyl. In optional embodiments, R2 is selected from methyl.

[0128] In optional embodiments, R1 is selected from halogen or optionally substituted C1-C6 alkoxy, and R2 is selected from optionally substituted C1-C6 alkyl. In optional embodiments, R1 is selected from fluorine or methoxy, and R2 is selected from methyl.

[0129] In optional embodiments, the [2'-R1-2'-R2] modified nucleotide is selected from one or both of [2'-F-2'-CH3] modified nucleotide and [2'-OCH3-2'-CH3] modified nucleotide. In optional embodiments, the sense strand is 17-33 nt in length, for example can be but not limited to 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nt.

[0130] The antisense strand is 18-35 nt in length, for example can be but not limited to 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nt.

[0131] In optional embodiments, the double-stranded oligonucleotide is selected from siRNA.

[0132] In optional embodiments, the double-stranded oligonucleotide has a structure represented by Formula (I):

[0133] wherein SS represents the sense strand, and AS represents the antisense strand.

[0134] o represents a phosphodiester internucleoside linkage, and s represents a phosphorothioate internucleoside linkage.

[0135] N1 represents an unmodified or modified nucleotide, and b1 represents the number of nucleotides N1, b1 is selected from an integer from 2 to 4 (e.g., 2, 3, or 4), and adjacent two N1 are connected by a phosphorothioate linkage. In optional embodiments, b1 is selected from 3.

[0136] N1' represents an unmodified or modified nucleotide, and b2 represents the number of nucleotides N1', b2 is selected from an integer from 2 to 4 (e.g., 2, 3, or 4), and adjacent two N1' are connected by a phosphodiester linkage or a phosphorothioate linkage. In optional embodiments, b2 is selected from 2. In optional embodiments, adjacent two N1' are connected by a phosphodiester linkage.

[0137] N x represents an unmodified or modified nucleotide, and c1 represents the number of nucleotides N x , c1 is selected from an integer from 12 to 19 (e.g., 12, 13, 14, 15, 16, 17, 18, or 19), and adjacent two N x are connected by a phosphodiester linkage or a phosphorothioate linkage.

[0138] N x ' represents an unmodified or modified nucleotide, and c2 represents the number of nucleotides N x ', c2 is selected from an integer from 12 to 19 (e.g., 12, 13, 14, 15, 16, 17, 18, or 19), and adjacent two Nx' are connected by a phosphodiester linkage or a phosphorothioate linkage, and there is at least one phosphorothioate linkage in the

[0139] N2 represents an unmodified or modified nucleotide, and d1 represents the number of nucleotides N2, d1 is selected from an integer from 2 to 4 (e.g., 2, 3, or 4), and adjacent two N2 are connected by a phosphodiester linkage or a phosphorothioate linkage. In optional embodiments, d1 is selected from 2 or 3. In optional embodiments, d1 is selected from 2. In optional embodiments, adjacent two N2 are connected by a phosphorothioate linkage.

[0140] N2' represents an unmodified or modified nucleotide, d2 represents the number of nucleotides N2', d2 is selected from an integer from 2 to 4 (e.g. 2, 3 or 4), and the two adjacent N2' are linked by phosphorothioate linkages.

[0141] B1 is selected from a [2'-R1-2'-R2] modified nucleotide, a1 represents the number of nucleotides N2', a1 is selected from an integer from 0 to 3 (e.g. 0, 1, 2 or 3).

[0142] B2 is selected from a [2'-R1-2'-R2] modified nucleotide, e1 represents the number of nucleotides N2', e1 is an integer from 0 to 3 (e.g. 0, 1, 2 or 3).

[0143] B3 is selected from a [2'-R1-2'-R2] modified nucleotide, a2 represents the number of nucleotides N2', a2 is an integer from 0 to 3 (e.g. 0, 1, 2 or 3).

[0144] and at least one of a1, e1 and a2 in formula (I) is not 0:

[0145] in case a1 is not 0, represents an overhang at the 5' end of the sense strand;

[0146] in case e1 is not 0, represents an overhang at the 3' end of the sense strand;

[0147] in case a2 is not 0, represents an overhang at the 3' end of the antisense strand.

[0148] In an optional embodiment, a1 is selected from 0.

[0149] In an optional embodiment, e1 is selected from 0.

[0150] In an optional embodiment, a2 is selected from an integer from 1 to 3. In an optional embodiment, a2 is selected from 2 or 3. In an optional embodiment, a2 is selected from 2.

[0151] In an optional embodiment, a1 is selected from 0, e1 is selected from 0, and a2 is selected from 2.

[0152] In an optional embodiment, there is a phosphorothioate linkage between the 10th and the 1 1 th nucleotide of the antisense strand, counting from the 5' end;

[0153] and / or there is a phosphodiester linkage between the nucleotide of the sense strand which is base-pairing with the 10th nucleotide of the antisense strand and the nucleotide of the sense strand which is base-pairing with the 1 1 th nucleotide of the antisense strand, counting from the 5' end.

[0154] In optional embodiments, the double-stranded oligonucleotide has a structure represented by Formula (II):

[0155] wherein each o / s is independently a phosphodiester internucleosidic linkage or a phosphorothioate internucleosidic linkage;

[0156] j1is selected from 7;

[0157] j1'is selected from 6;

[0158] j2is selected from an integer from 5 to 7 (e.g., 5, 6, or 7), j2'is selected from an integer from 6 to 8 (e.g., 6, 7, or 8), and j2' = j2 + 1.

[0159] In optional embodiments, j2is selected from 5 and j2'is selected from 6, or j2is selected from 6 and j2'is selected from 7, or j2is selected from 7 and j2'is selected from 8.

[0160] In optional embodiments, j2is selected from 5 and j2'is selected from 6.

[0161] In optional embodiments, N1, N1', N2, N2', N x , and N x are each independently selected from modified nucleotides. N1, N1', N2, N2', N x , and N x may be the same modified nucleotide or different modified nucleotides.

[0162] In optional embodiments, the modified nucleotides are each independently selected from at least one of a 2'-halogen modified nucleotide, a 2'-deoxy modified nucleotide, a 2'-O-R3modified nucleotide, a 2'-O-(CH2) n -R4modified nucleotide, a [2'-F-2'-F] modified nucleotide, or a nucleotide analog; wherein the two F in the [2'-F-2'-F] modified nucleotide are substituent groups that replace the 2'-OH and 2'-H of the ribose ring of the nucleotide, respectively;

[0163] R3is selected from optionally substituted C1-C6alkyl;

[0164] n is selected from an integer from 0 to 3, and R4is selected from optionally substituted C1-C6alkoxy or R 4a , R 4b , and R 4c are each independently selected from optionally substituted C1-C6alkyl or optionally substituted C1-C6alkoxy;

[0165] The nucleotide analogs include one or more of a peptide nucleic acid (PNA), a morpholino nucleic acid (MNA), a bridged nucleic acid (BNA), a locked nucleic acid (LNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), or an unlocked nucleic acid (UNA).

[0166] In optional embodiments, the 2'-halogen modified nucleotide is selected from a 2'-F modified nucleotide.

[0167] In optional embodiments, the 2'-O-R3modified nucleotide is selected from a 2'-O-CH3modified nucleotide.

[0168] In optional embodiments, the 2'-O-(CH2) n -R4modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide, a 2'-O-TOM modified nucleotide, a 2'-O-CH2-O-CH2-CH3modified nucleotide, a 2'-O-CH2-O-CH2-CF3modified nucleotide, or a 2'-O-CH2-CH2-O-CH3modified nucleotide.

[0169] In optional embodiments, the 2'-O-(CH2) n -R4modified nucleotide is selected from a 2'-O-CH2-CH2-O-CH3.

[0170] In optional embodiments, the double-stranded oligonucleotide contains at least one 2'-O-(CH2) n -R4modified nucleotide.

[0171] In optional embodiments, the double-stranded oligonucleotide contains at least one 2'-O-(CH2) n -R4modified nucleotide; each of the 2'-O-(CH2) n -R4modified nucleotide is independently located at position 5, 7, 8, 10, 12, 18, 19 of the sense strand or position 8, 10, 15, 20, 21 of the antisense strand, counting from the 5' end.

[0172] In optional embodiments, the double-stranded oligonucleotide contains one 2'-O-(CH2) na nucleotide modified with -R4.

[0173] In optional embodiments, the double stranded oligonucleotide contains one 2'-0-(CH2) n a nucleotide modified with -R4, and the 2'-0-(CH2) n the nucleotide modified with -R4 is located at the 15th position from the 5' end of the antisense strand.

[0174] In optional embodiments, the double stranded oligonucleotide contains one 2'-0-(CH2) n a nucleotide modified with -R4, and the 2'-0-(CH2) n the nucleotide modified with -R4 is located at the 15th position from the 5' end of the antisense strand.

[0175] In optional embodiments, the double stranded oligonucleotide contains at least one [2'-F-2'-F] modified nucleotide in the duplex region.

[0176] In optional embodiments, the double stranded oligonucleotide contains at least one [2'-F-2'-F] modified nucleotide in the duplex region; and at least one of the nucleotides at positions 2, 6, 9, 12, 14, 16 of the antisense strand and the nucleotides in the sense strand that base pair with the nucleotides at positions 10, 11, 12, 13, 15 of the antisense strand, counting from the 5' end of the antisense strand, is a [2'-F-2'-F] modified nucleotide.

[0177] In optional embodiments, the double stranded oligonucleotide contains one [2'-F-2'-F] modified nucleotide in the duplex region; and the [2'-F-2'-F] modified nucleotide is located at any of the positions 2, 6, 9, 12, 14, 16 of the antisense strand and the nucleotides in the sense strand that base pair with the nucleotides at positions 10, 11, 12, 13, 15 of the antisense strand, counting from the 5' end of the antisense strand.

[0178] In optional embodiments, the double stranded oligonucleotide contains one [2'-F-2'-F] modified nucleotide in the duplex region, and the [2'-F-2'-F] modified nucleotide is located at any of the positions 2, 6, 9, 12, 14, 16 of the antisense strand.

[0179] In optional embodiments, the double stranded oligonucleotide contains one [2'-F-2'-F] modified nucleotide in the duplex region, and the [2'-F-2'-F] modified nucleotide is located at the 14th position of the antisense strand, counting from the 5' end of the antisense strand.

[0180] In optional embodiments, the sense strand has 19 nucleotides, and the antisense strand has 21 nucleotides;

[0181] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides;

[0182] and / or, at least three of the 7th-10th nucleotides in the sense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0183] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 14th nucleotide is selected from [2'-F-2'-F], the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides;

[0184] and / or, at least three of the 7th-10th nucleotides in the sense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0185] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 15th nucleotide is selected from 2'-O-CH2-CH2-O-CH3 modified nucleotides, the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides;

[0186] and / or, at least three of the 7th-10th nucleotides in the sense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0187] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th, 20th, and 21st nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 14th nucleotide is selected from [2'-F-2'-F], the 15th nucleotide is selected from a 2'-O-CH2-CH2-O-CH3 modified nucleotide, the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0188] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th, 20th, and 21st nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 14th nucleotide is selected from [2'-F-2'-F], the 15th nucleotide is selected from a 2'-O-CH2-CH2-O-CH3 modified nucleotide, the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0189] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th, 20th, and 21st nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 14th nucleotide is selected from [2'-F-2'-F], the 15th nucleotide is selected from a 2'-O-CH2-CH2-O-CH3 modified nucleotide, the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0190] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th, 20th, and 21st nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 14th nucleotide is selected from [2'-F-2'-F], the 15th nucleotide is selected from a 2'-O-CH2-CH2-O-CH3 modified nucleotide, the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0191] In optional embodiments, at least four of the 2nd, 6th, 9th, 12th, 14th, 16th, 20th, and 21st nucleotides in the antisense strand, in the 5' to 3' direction, are selected from 2'-F modified nucleotides, the 14th nucleotide is selected from [2'-F-2'-F], the 15th nucleotide is selected from a 2'-O-CH2-CH2-O-CH3 modified nucleotide, the 20th-21st nucleotides are selected from [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotides, and the remaining nucleotides are selected from 2'-O-CH3 modified nucleotides.

[0192] In an alternative embodiment, the 2nd, 6th, 9th, 14th, 16thposition or the 2nd, 6th, 12th, 14th, 16thposition in the antisense strand is selected from a 2'-F modified nucleotide, the 15thposition is selected from a 2'-O-CH2-CH2-O-CH3modified nucleotide, the 20th-21stposition is selected from a [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotide, and the remaining positions are selected from 2'-O-CH3modified nucleotides, in the 5' end to 3' end direction;

[0193] and / or, the 7th-10thposition in the sense strand is selected from a 2'-F modified nucleotide, and the remaining positions are selected from 2'-O-CH3modified nucleotides, in the 5' end to 3' end direction.

[0194] In an alternative embodiment, the 2nd, 6th, 9th, 14th, 16thposition or the 2nd, 6th, 12th, 14th, 16thposition in the antisense strand is selected from a 2'-F modified nucleotide, the 14thposition is selected from [2'-F-2'-F], the 15thposition is selected from a 2'-O-CH2-CH2-O-CH3modified nucleotide, the 20th-21stposition is selected from a [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotide, and the remaining positions are selected from 2'-O-CH3modified nucleotides, in the 5' end to 3' end direction;

[0195] and / or, the 7th-10thposition in the sense strand is selected from a 2'-F modified nucleotide, and the remaining positions are selected from 2'-O-CH3modified nucleotides, in the 5' end to 3' end direction.

[0196] In an alternative embodiment, the double-stranded oligonucleotide is selected from an siRNA.

[0197] Conjugate:

[0198] In a second aspect, the present disclosure provides a conjugate comprising the double-stranded oligonucleotide of the first aspect and one or more targeted delivery ligands.

[0199] In an alternative embodiment, the targeted delivery ligand is selected from a liver-targeted delivery ligand, a kidney-targeted delivery ligand, a lung-targeted delivery ligand, a muscle-targeted delivery ligand, a fat-targeted delivery ligand, a brain-targeted delivery ligand, a heart-targeted delivery ligand, or a CNS-targeted delivery ligand.

[0200] In optional embodiments, the targeting delivery ligand is conjugated to the antisense strand and / or the sense strand. In optional 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 conjugated to the targeting delivery ligand.

[0201] In optional embodiments, the targeting delivery ligand is selected from 1, 2, 3, or 4.

[0202] In optional embodiments, the targeting delivery ligand is 1. Wherein one of the targeting delivery ligand 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.

[0203] In optional embodiments, the targeting delivery ligand is 1, one of the targeting delivery ligand is conjugated to the 3' end of the sense strand.

[0204] In optional embodiments, the number of targeting delivery ligands is selected from 1.

[0205] In optional embodiments, the number of targeting delivery ligands is selected from 1, one of the ligands is conjugated to the 3' end of the sense strand.

[0206] In optional embodiments, the targeting delivery ligand is selected from the structure of Formula (CR01008x3), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0207] In optional embodiments, the targeting delivery ligand is selected from the structure of Formula (CR01008x4), or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0208] In optional embodiments, the targeting delivery ligand is selected from the structure of Formula L96, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.

[0209] Compositions:

[0210] In a third aspect, the present disclosure provides a composition comprising the conjugate of the second aspect.

[0211] Uses:

[0212] In a fourth aspect, the present disclosure provides use of any of the following in the manufacture of a medicament for preventing and / or treating a disease:

[0213] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0214] (II) the conjugate of the second aspect; and / or

[0215] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0216] In optional embodiments, the disease is a disease or condition mediated by the target gene.

[0217] In optional embodiments, the disease is a disease or condition associated with expression of the target gene.

[0218] Use:

[0219] In a fifth aspect, the disclosure provides use of any of the following in the manufacture of a medicament for reducing expression or activity of a target gene:

[0220] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0221] (II) the conjugate of the second aspect; and / or

[0222] (III) the composition of the third aspect.

[0223] Pharmaceutical composition:

[0224] In a sixth aspect, the disclosure provides a pharmaceutical composition comprising any of the following and a pharmaceutically acceptable excipient:

[0225] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0226] (II) the conjugate of the second aspect; and / or

[0227] (III) the composition of the third aspect.

[0228] Method:

[0229] In a seventh aspect, the disclosure provides a method of reducing expression or activity of a target gene, the method comprising contacting a cell with any of the following:

[0230] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0231] (II) the conjugate of the second aspect; and / or

[0232] (III) the composition of the third aspect; and / or

[0233] (IV) the pharmaceutical composition of the sixth aspect.

[0234] In optional embodiments, the double-stranded oligonucleotide of the first aspect, the conjugate of the second aspect, the composition of the third aspect, or the pharmaceutical composition of the sixth aspect is administered in a buffered solution or a non-buffered solution.

[0235] In optional embodiments, the inhibition of the target gene expression is 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 the target gene expression can be determined in a conventional manner known in the art, for example, but not limited to, the inhibition rate determined by luciferase reporter assay, PCR method, or protein-based method, such as immunofluorescence analysis, for example, Western Blot or flow cytometry.

[0236] In optional embodiments, the double-stranded oligonucleotide described above, and / or the conjugate described above is administered at a dose of 0.01 mg / kg to 10 mg / kg, or 0.5 mg / kg to 50 mg / kg.

[0237] In optional embodiments, the double-stranded oligonucleotide described above, and / or the conjugate described above is administered subcutaneously or intravenously.

[0238] The present disclosure is further illustrated by specific examples below, but it should be understood that these examples are merely used for more detailed illustration, and should not be understood as limiting the present disclosure in any form.

[0239] Unless otherwise specified, the siRNA sequences used in the present disclosure are synthesized by Suzhou Xuanjing Biotechnology Co., Ltd. and Suzhou Baisin Biotechnology Co., Ltd.; the PCR primers used in the present disclosure are synthesized by Beijing Qikexin Biotechnology Co., Ltd.; and the experimental animals C57BL / 6J mice used in the present disclosure are purchased from Spafas (Beijing) Biotechnology Co., Ltd.

[0240] Unless otherwise specified, the reagents, consumables and instruments used in the present disclosure are all from commercially available products. Among them, the main reagents and consumables are shown in Table 1, and the main instruments are shown in Table 2.

[0241] Table 1 Main reagents and consumables

[0242] Table 2 Main instruments

[0243] Preparation Example 1: Synthesis of compound NM054

[0244] In the present preparation example, the synthesis route of compound NM054 is as follows:

[0245] (1-1) Synthesis of compound NM054-2

[0246] Into a 500 ml reaction kettle, compound NM054-1 (3 g, 11.54 mmol, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methyl uridine, CAS No. 863329-66-2) and pyridine (30 ml) were added, and the temperature was lowered to 0°C. 4,4'-Dimethoxytrityl chloride (4.29 g, 12.7 mmol, 1.1 eq) was added in portions, and the reaction system was stirred at 25°C under a nitrogen atmosphere for 3 hours. HPLC showed that there was no starting material. After the reaction was completed, the reaction solution was concentrated, and purified water (50 ml) and ethyl acetate (50 ml) were added for extraction. The organic phase was separated, dried over anhydrous sodium sulfate, and filtered and concentrated. Purification by column chromatography gave compound NM054-2 (2.7 g, yield 41.7%). MS ESI (m / z) = 563.0 [M+H] + .

[0247] Synthesis of compound NM054

[0248] Into a 100 ml reaction kettle, compound NM054-2 (2.7 g, 4.8 mmol, 1.0 eq) was added, and bis(diisopropylamino)(2-cyanoethoxy) phosphine (1.74 g, 5.76 mmol, 1.2 eq) was added in portions. Then 4,5-dicyanoimidazole (0.45 g, 3.8 mmol, 0.8 eq, abbreviated as DCI, CAS No. 1122-28-7) and dichloromethane (27 ml) were added, and the reaction system was stirred at 25°C under a nitrogen atmosphere for 3 hours. After the reaction was completed, sodium bicarbonate aqueous solution (20 ml) was added to the reaction solution, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to give compound NM054 (3.0 g). MS ESI (m / z) = 763 [M+H] + .

[0249] Preparation Example 2: Preparation of compound NM118

[0250] Synthesis of compound NM118-2

[0251] Compound NM118-1 (20 g, 1.0 eq, 2'-C-methyluridine, CAS No. 31448-54-1) was dissolved in pyridine (100 ml), 1,3-dichloro-1,1,3,3-tetraisopropyl disiloxane (28.6 g, 1.2 eq, Alias TiPDSC12, CAS No. 69304-37-6) was added, stirred at room temperature for 12 hours, and the reaction was completed. The reaction solution was concentrated, water (100 ml) was added, and extracted with ethyl acetate twice (100 ml each time), the organic phase was dried and concentrated, and purified by normal phase column chromatography (eluent: ethyl acetate / petroleum ether = 34 / 66, v / v) to obtain compound NM118-2 (39 g) as a white solid. MS ESI (m / z) = 501 [M+H] + .

[0252] (2-2) Synthesis of compound NM118-3

[0253] Compound NM118-2 (5.0 g, 1 eq) was dissolved in anhydrous tetrahydrofuran (50 ml), and a 60 wt% NaH (1.6 g, 4 eq) anhydrous THF solution was added under ice bath, and nitrogen was replaced 3 times. The reaction system was stirred under ice bath for 1 hour under nitrogen atmosphere, and iodomethane (3 g, 2 eq) was added, and reacted at room temperature for 4 hours, and the reaction was completed. Saturated aqueous ammonium chloride solution (20 ml) was added to the reaction solution, and extracted with ethyl acetate 3 times (20 ml each time), and the organic phase was combined, dried and concentrated, and purified by normal phase column chromatography (eluent: acetonitrile / water = 25 / 75, v / v) to obtain compound NM118-3 (3 g) as a white powder. MS ESI (m / z) = 515 [M+H] + .

[0254] (2-3) Synthesis of compound NM118-4

[0255] Compound NM118-3 (3.0 g, 1 eq) was dissolved in tetrahydrofuran (30 ml), and a 1M tetrabutylammonium fluoride (TBAF, CAS No. 429-41-4) tetrahydrofuran solution (3 ml, 0.5 eq) was added, and stirred at room temperature for 1 hour, and the reaction was completed. Water (20 ml) was added to the reaction solution, and extracted with ethyl acetate 3 times (20 ml each time), and the organic phase was combined, dried and concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 36 / 64, v / v) to obtain compound NM118-4 (1.1 g) as a white powder. MS ESI (m / z) = 295 [M+Na] + .

[0256] (2-4) Synthesis of compound NM118-5

[0257] Compound NM118-4 (1.1 g, 1.0 eq) was dissolved in pyridine (20 ml), 4,4'-dimethoxytrityl chloride (1.7 g, 1.3 eq, referred to as DMTrCl, CAS No. 40615-36-9) was added under ice-bath, the reaction system was stirred at room temperature for 3 hours under nitrogen atmosphere, methanol (20 ml) was added to quench the reaction, and the reaction was completed. The reaction solution was concentrated, water (20 ml) was added, and the organic phase was extracted with ethyl acetate for 3 times (20 ml each time), the organic phase was combined, dried and concentrated, and purified by normal phase column chromatography (eluent: petroleum ether / ethyl acetate = 50 / 50, v / v) to obtain compound NM118-5 (1.8 g) in the form of light yellow solid. MS ESI (m / z) = 575 [M+H] + .

[0258] Synthesis of compound NM118-6

[0259] Compound NM118-5 (1.6 g, 1.0 eq) was dissolved in anhydrous dichloromethane (20 ml), 4,5-dicyanoimidazole (264.4 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy) phosphine (0.92 g, 1.1 eq) were added respectively, the reaction system was stirred at room temperature for 2 hours under nitrogen atmosphere, and the reaction was completed. Saturated aqueous sodium bicarbonate solution (20 ml) was added to the reaction solution, and the organic phase was extracted with dichloromethane for 3 times (20 ml each time), the organic phase was combined, dried and concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 75 / 25, v / v) to obtain compound NM-118 (1.7 g, yield 78.7%) in the form of white powder. MS ESI (m / z) = 775 [M+H] + .

[0260] 1 H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 7.99 (dd, J = 8.1, 1.6 Hz, 1H), 7.44 - 7.18 (m, 9H), 6.92 (ddd, J = 10.7, 7.4, 3.5 Hz, 4H), 5.96 (d, J = 9.5 Hz, 1H), 5.77 (s, 1H), 4.97 (t, J = 8.7 Hz, 1H), 4.38 - 4.07 (m, 2H), 3.76 (d, J = 3.1 Hz, 7H), 3.50 (ddt, J = 13.9, 11.6, 7.1 Hz, 4H), 3.40 (d, J = 6.7 Hz, 3H), 2.81 (t, J = 5.8 Hz, 1H), 2.64 - 2.54 (m, 1H), 1.25 (d, J = 3.3 Hz, 3H), 1.19 - 1.03 (m, 10H), 0.92 (d, J = 6.7 Hz, 2H).

[0261] Preparation Example 3: Preparation of compound NM131

[0262] The structural formula of compound NM131 is

[0263] In this preparation example, the synthetic route of compound NM131 is as follows:

[0264] (1-1) Synthesis of compound NM131-2.

[0265] Compound NM131-1 (2 g, 7.58 mmol, 1 eq, CAS No. 114248-23-6) was dissolved in pyridine (20 mL) at room temperature, and the solution was cooled to 0 °C in an ice bath. Then, DMTrCl (3.33 g, 9.85 mmol, 1.3 eq) was added portionwise. The reaction solution was stirred at 25 °C for 1 h. After the reaction was completed, methanol was added to quench the reaction, and the solvent was removed by rotary evaporation. The residue was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (2 x 30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, and filtered and concentrated. The residue was 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] + .

[0266] (1-2) Synthesis of compound NM131

[0267] Compound NM131-2 (2.5 g, 4.41 mmol, 1 eq) that had been dried by azeotropic dehydration with acetonitrile three times (30 mL each time) was dissolved in dichloromethane (25 mL). Then, a solution of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.99 g, 6.62 mmol, 1.5 eq) in dichloromethane (25 mL) that had been dried by azeotropic dehydration with acetonitrile three times (30 mL each time) was added, followed by the addition of 1H-imidazole-4,5-dicarbonitrile (416.3 mg, 3.53 mmol, 0.8 eq). The reaction solution was stirred at 25 °C for 1 h under a nitrogen atmosphere. After the reaction was completed, the reaction solution was diluted with saturated aqueous sodium bicarbonate solution (50 mL), and the mixture was extracted with dichloromethane (3 x 30 mL). The combined organic phase was washed with saturated brine (2 x 30 mL), dried over anhydrous sodium sulfate, and filtered and concentrated. The residue was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 2 / 1) to give compound NM131 (2.86 g, yield 84.62%) as a white solid. MS ESI (m / z) = 767.4 [M+H] + .

[0268] Preparation Example 4: Preparation of compound CR01008 and compound CR01008Z

[0269] In this preparation example, the synthesis route of compound CR01008 and compound CR01008Z is as follows:

[0270] (4-1) Synthesis of compound CR01008-2

[0271] Compound CR01008-1 (10.0 g, 1.0 eq, trans-4-(Boc-amino)cyclohexylcarboxaldehyde, CAS No. 181308-57-6) and a 37 wt% aqueous formaldehyde solution (8.9 g, 2.4 eq) were dissolved in methanol (33 ml), and a 45.3 wt% aqueous KOH solution (13 ml) was added dropwise. After the dropwise addition was completed, the reaction was stirred at 25°C for 30 minutes, the temperature was raised to 60°C, and the reaction was refluxed at 60°C for 2 hours. The reaction was completed. After the reaction solution was cooled to room temperature, the reaction solution was evaporated under reduced pressure to obtain a crude product in the form of a white solid. A small amount of water was added to the crude product to make a slurry, and filtration was performed to obtain compound CR01008-2 (9 g, yield 78.9%) in the form of a white solid. MS-ESI (m / z) = 260 [M+H] + .

[0272] (4-2) Synthesis of compound CR01008-3

[0273] Compound CR01008-2 (9 g, 1 eq) was dissolved in 1,4-dioxane (70 ml), and a 4M hydrogen chloride solution in 1,4-dioxane (45 ml) was added. The reaction was stirred at 25°C for 1 hour, and the reaction was completed. The reaction solution was evaporated under reduced pressure to obtain compound CR01008-3 (6.8 g, yield 100%) in the form of a white solid.

[0274] (4-3) Synthesis of compound CR01008-5

[0275] Compound CR01008-3 (1.8 g, 2.0 eq), compound CR01008-4 (2.1 g, 1.0 eq, 5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, CAS No. 1159408-54-4) and N,N-diisopropylethylamine (3.5 g, 6.0 eq, referred to as DIEA, CAS No. 7087-68-5) were dissolved in DMF (15 ml), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.9 g, 1.1 eq, referred to as HBTU, CAS No. 94790-37-1) was added, the reaction system was stirred at 25 °C for 3 hours under nitrogen atmosphere, and the reaction was completed. The reaction solution was evaporated under reduced pressure, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 22 / 78, v / v) to obtain compound CR01008-5 (1.78 g, yield 64.4%) in the form of a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0276] Synthesis of compound CR01008-6

[0277] Compound CR01008-5 (1.54 g, 1.0 eq) was dissolved in pyridine (15 ml), the reaction system was cooled to 0 °C with an ice water bath, and DMTrCl (1.32 g, 1.5 eq) was added at 0 °C, and the reaction was carried out at 25 °C for 3 hours. Methanol (15 ml) was added to quench the reaction, and the reaction was completed. The reaction solution was evaporated under reduced pressure, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 60 / 40, v / v) to obtain compound CR01008-6 (1 g, yield 42.7%) in the form of a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0278] Synthesis of compound CR01008

[0279] Compound CR01008-6 (1.08 g, 1.0 eq) was dissolved in anhydrous dichloromethane (20 ml), and DCI (115 mg, 0.8 eq) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (732 mg, 2.1 eq) were added, respectively. The reaction system was stirred for 2 hours at 25 °C under nitrogen atmosphere after three times of nitrogen replacement. After the reaction was completed, saturated aqueous sodium bicarbonate solution (20 ml) was added to the reaction solution, and dichloromethane was extracted three times (20 ml each time). The organic phase was combined, dried, and concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 72 / 28, v / v) to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0280] 1H NMR (400 MHz, DMSO-d6) δ 1.05 (d, J = 6.7 Hz, 6H), 1.14 (d, J = 6.7 Hz, 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 = 5.9 Hz, 2H), 2.79 (d, J = 8.4 Hz, 1H), 2.87 (d, J = 8.4 Hz, 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, 8.8 Hz, 1H), 4.05 (s, 3H), 4.51 (d, J = 8.4 Hz, 1H), 4.99 (dd, J = 11.3, 3.4 Hz, 1H), 5.24 (d, J = 3.4 Hz, 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 = 7.8 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H).

[0281] (4-6) Synthesis of compound CR01008-7

[0282] Compound CR01008-6 (500 mg) was dissolved in dichloromethane (10 ml), succinic anhydride (112 mg), 4-dimethylaminopyridine (6.8 mg, abbreviated as DMAP) and triethylamine (226.2 mg) were added, and the reaction system was stirred at 25°C under nitrogen atmosphere for 16 hours after nitrogen replacement for 3 times. The reaction was completed. The reaction solution was purified by FLASH to obtain compound CR01008-7 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0283] Synthesis of compound CR01008Z

[0284] A 20-ml sample bottle was added with compound CR01008-7 (50 mg), amino CPG (1.25 g, 0.1 mmol), HBTU (27 mg), DIEA (12 mg), and the reaction was carried out on a shaking table for 16 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was first washed with acetonitrile (10 ml) once and then dried under vacuum. A 20-ml sample bottle was added with the dried filter cake, 4-dimethylaminopyridine (3 mg, abbreviated as DMAP, CAS number 1122-58-3), Cap1 (10 ml) and Cap2 (1 ml), and the reaction was carried out on a shaking table for 6 hours. After the reaction was completed, the reaction solution was filtered, and the filter cake was first washed with 10 ml of acetonitrile once and then dried under vacuum to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0285] The structural formula of the amino CPG (Aminoalkyl-CPG) is as follows: Model C3006-1000, 100-200 mesh, loading 80 μmol / g.

[0286] Cap1 and Cap2 are cap reagents, Cap1 is a 20% (by volume) N-methylimidazole solution in pyridine / acetonitrile, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is a 20% (by volume) acetic anhydride solution in acetonitrile.

[0287] Preparation Example 5: Preparation of siRNA conjugate

[0288] Compound L96-PS was purchased from Kalley Medicine Group (Tianjin) Co., Ltd., and the loading was 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is as follows:

[0289] PS represents a polystyrene (Polystyrene) resin solid phase carrier.

[0290] The structural formula of compound NM130 is as follows: Obtained commercially.

[0291] (5-1) Synthesis of sense strand (SS) and antisense strand (AS)

[0292] By the method of phosphoramidite nucleic acid solid-phase synthesis, starting with the above-mentioned compound linked to a solid-phase carrier (for example, CPG carrier, PS carrier, compound L96-PS, compound CR01008Z), nucleoside monomers are sequentially linked in the order of 3'-5' according to the nucleotide sequence. During the synthesis, compounds NM054, NM118, NM130, NM131, CR01008 are regarded as one nucleoside monomer, respectively.

[0293] Each nucleoside monomer linkage includes four-step reactions of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0294] The nucleoside monomers are prepared into an acetonitrile solution of nucleoside monomers with a concentration of 0.1 M.

[0295] The conditions of deprotection reaction in each step are the same. The conditions of deprotection reaction are as follows: temperature is 25°C, reaction time is 70 seconds, deprotection reagent is dichloroacetic acid in dichloromethane solution (3% by volume), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid-phase carrier is 5:1.

[0296] The conditions of coupling reaction in each step are the same. The conditions of coupling reaction are as follows: temperature is 25°C, the molar ratio of the nucleic acid sequence linked on the solid-phase carrier to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence linked on the solid-phase carrier to the coupling reagent is 1:65, reaction time is 600 seconds, the coupling reagent is 5-ethylthio-1H-tetrazole in acetonitrile solution with a concentration of 0.5 M, and the sulfur reagent is a mixture of acetonitrile / pyridine with a volume ratio of 1:1, in which the concentration of hydrogenated xanthine is 0.2 mol / L.

[0297] The conditions of capping reaction in each step are the same. The conditions of capping reaction are as follows: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixture of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is N-methylimidazole in pyridine / acetonitrile mixed solution with a concentration of 20% by volume, the volume ratio of pyridine to acetonitrile is 3:5, and Cap2 is acetic anhydride in acetonitrile solution with a concentration of 20% by volume; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent, and the nucleic acid sequence linked on the solid-phase carrier is 1:1:1.

[0298] The conditions for each oxidation reaction are the same. The conditions for the oxidation reaction are: temperature is 25°C; reaction time is 3 seconds; concentration of the oxidizing agent is 0.05 M iodine water, molar ratio of iodine to the nucleic acid sequence attached to the solid support in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine is 1:9). The conditions for the sulfurization reaction are: temperature is 25°C; reaction time is 360 seconds; concentration of the sulfurizing agent is 0.2 M pyridine solution of hydrogen xanthate, molar ratio of the sulfurizing agent to the nucleic acid sequence attached to the solid support in the coupling reaction is 4:1; the sulfurization reaction is carried out in a water / pyridine mixed solvent (volume ratio of water to pyridine is 1:9).

[0299] After the last nucleoside monomer is attached, the nucleic acid sequence attached to the solid support is subjected to cleavage, deprotection, purification, desalting, and then lyophilization in sequence to obtain the sense strand, wherein:

[0300] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence attached to the solid support is added to 25% ammonia water by mass, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and vacuum concentration is carried out to dryness. After ammonia water treatment, 0.4 ml / μmol of N-methylpyrrolidine is used to dissolve the product relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trifluoromethanesulfonate to remove the 2'-O-TBDMS protection on the ribose.

[0301] The purification and desalting conditions are as follows: the purification of the nucleic acid is completed by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 is 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (volume ratio of water to acetonitrile is 9:1); eluent 2 is 1.5 M sodium chloride, 20 mM sodium phosphate (pH=8.1), the solvent is a water / acetonitrile mixed solution (volume ratio of water to acetonitrile is 9:1); the elution gradient is eluent 1:eluent 2=(100:0)-(50:50). After the product eluate is collected, desalting is carried out using a reverse phase chromatography purification column, and the desalting conditions include desalting using a dextran gel column, the filler is dextran gel G25, and elution is carried out using deionized water.

[0302] Detection: purity detection is carried out using ion exchange chromatography (IEX-HPLC); molecular weight detection is carried out using liquid chromatography-mass spectrometry (LC-MS), and the actual value of the molecular weight is compared with the theoretical value; if the actual value and the theoretical value are consistent, it is indicated that the sense strand and the target antisense strand are obtained.

[0303] (4-2) Synthesis of siRNA conjugates

[0304] The sense strand and the antisense strand synthesized in step (4-1) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95°C, slowly cooled to room temperature and kept at room temperature for 10 minutes, and the sense strand and the antisense strand were allowed to form a double-stranded structure by hydrogen bonding, thereby obtaining an siRNA conjugate having the sense strand and the antisense strand shown in Table 3.

[0305] wherein, when the delivery carrier is L96, the structural formula of the siRNA conjugate is:

[0306] When the delivery carrier is a tri-cluster CR01008 (denoted as: (CR01008Z)(CR01008)(CR01008) or (CR01008x3)), the structural formula of the siRNA conjugate is:

[0307] When the delivery carrier is a tetra-cluster CR01008 (denoted as: (CR01008Z)(CR01008)(CR01008)(CR01008) or (CR01008x4)), the structural formula of the siRNA conjugate is:

[0308] wherein, denotes siRNA.

[0309] Table 3 Sequence information of siRNA conjugate

[0310] Table 4 Molecular weight information of siRNA conjugate

[0311] Method for evaluating the inhibitory activity of siRNA conjugate on target gene on mouse primary liver

[0312] Isolation of mouse primary hepatocytes:

[0313] The mouse primary hepatocytes were extracted from the fresh liver tissue of C56BL / 6j mice. The specific operation steps were as follows: the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate solution, and the mice were fixed and the abdomen and chest were disinfected with 75% ethanol. The surgical instruments were sterilized, the hepatic portal vein and inferior vena cava were exposed by opening the abdominal cavity. The heparin cap was installed on the needle, and the scalp needle connected to the infusion pump bottle (0.5 mM EDTA HBSS perfusion solution) was connected, the needle was inserted into the inferior vena cava, and the perfusion was performed at a rate of 120 drops / min. The hepatic portal vein was cut to make the perfusion solution flow out of the cut hepatic portal vein, and the perfusion was continued for 4 minutes, and then 0.8 mg / mL collagenase type IV HBSS solution (Sigma, C5138) (containing 0.08% DNA I enzyme (sigma, DN25)) was replaced to continue perfusion for 8 minutes. The perfused liver was removed from the animal, washed with HBSS (containing Ca2+, Mg2+, MACGENE, CC016), placed in a sterile culture dish, added with DMEM complete medium (DMEM medium + 10% serum), and the liver was torn apart. The cell suspension was filtered through a cell sieve to remove undigested tissues and connective tissues, centrifuged at 800 rpm for 3 min to discard the supernatant, and the DMEM complete medium was added again to suspend and centrifuge to obtain the primary mouse hepatocytes.

[0314] Cell culture and transfection:

[0315] The DMEM complete medium was added to adjust the cell density to 2×10 5 cells / mL to obtain a mouse primary hepatocyte suspension. Then the cells were inoculated into a 12-well culture plate coated with mouse tail collagen type I (coating method according to the description of solarbio (C8062) at a concentration of 2 μg / cm 2 ) coated in advance. The volume of the added cell suspension was 1000 μL / well, and the amount of cells was 2×10 5 cells / well.

[0316] Each group of conjugates was diluted with PBS to a working solution (calculated by siRNA) of 1000 times the final test concentration. 1 μL / well of siRNA conjugate working solution was added to the above-mentioned 12-well culture plate, and 2-3 culture wells were set for each siRNA conjugate. Another 2-3 culture wells were added with 1 μL / well of PBS as blank control wells. Shake the culture plate to mix evenly. The culture plate was placed in a cell incubator at 37°C, 5% CO2 for continuous culture for 24 h.

[0317] RNA extraction: The total RNA of each group of primary hepatocyte samples was extracted using the automatic nucleic acid extractor and nucleic acid extraction kit of Zhejiang Hanwei Science and Technology Co., Ltd. according to the method described in the instruction manual.

[0318] Evaluation method of siRNA conjugate target gene inhibition activity in mice

[0319] 6-8 weeks old C57BL / 6J mice were randomly grouped by weight (all female). The mice in each group were calculated the dose of the drug according to the weight, and were given a single dose by abdominal subcutaneous injection, and each siRNA conjugate was prepared into a solution of the corresponding concentration (calculated by siRNA) with PBS solution for administration, and the administration volume was 5 ml (calculated by siRNA) / kg (calculated by mice). The PBS control group was given 5 ml / kg (calculated by mice) of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as DO), and at the preset time after administration, 5 mice were sacrificed in each group. The necropsy of the sacrificed mice was performed and the liver tissue of each sacrificed mouse was collected, and the liver tissue was cut into about 2 mm 3 pieces and stored in RNA later.

[0320] The liver tissue samples at different time points in different experimental groups were taken from the above RNA later, and the liver tissue samples were broken for 60 s in a Tissuelyser II type automatic tissue homogenizer, and then total RNA was extracted using a full-automatic 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 operation procedure for total RNA extraction.

[0321] mRNA expression level detection:

[0322] Take the above 1 μg total RNA, use the reverse transcription kit (Promega Company, Reverse Transcription System, A3500) and select Oligo(dT)15 reverse transcription primer, configure 20 μL reverse transcription system according to the method recorded in the reverse transcription kit instruction book and complete the reverse transcription reaction. After the reaction, 80 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. Then use real-time fluorescent quantitative PCR kit (ABI Company, SYBR TM Select Master Mix, Catalog number: 4472908) to detect the expression amount of target gene mRNA. In this real-time fluorescent quantitative PCR method, primers for target genes and primers for internal reference genes are used to detect target genes and internal reference genes, respectively. Configure 20 μL Real-time PCR reaction system for each PCR detection hole according to the method recorded in the real-time fluorescent quantitative PCR kit instruction book, and each reaction system contains 5 μL cDNA solution obtained by the above reverse transcription reaction, 10 μL SYBR TMSelect Master Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, 4 μL RNase-Free H2O. The prepared reaction system was placed on a real-time fluorescence quantitative PCR instrument (ABI Company, StepOnePlus TM ) and Real-time PCR amplification was performed using a three-step method. The amplification program was 95°C pre-denaturation for 10 min, followed by 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, and repeating the denaturation, annealing and extension processes for 40 cycles.

[0323] Alternatively, a reverse transcription kit (Thermo Fisher Scientific Company, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and an Oligo(dT)18 reverse transcription primer was selected. A 20 μL reverse transcription system was prepared according to the method recorded in the reverse transcription kit instruction book and the reverse transcription reaction was completed. Then, a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific Company, TaqMan Fast Advanced Master Mix, 4444557) was used to detect the expression amount of the target gene mRNA on a fluorescence quantitative PCR instrument (Bio-Rad Company, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and the primers for the target gene and the primers for the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively.

[0324] In this real-time fluorescence quantitative PCR method, the ΔΔCt method was used to calculate the expression level and inhibition rate of the target gene mRNA in each test group. The calculation method was as follows:

[0325] ΔCt(test group) = Ct(test group target gene) - Ct(test group internal reference gene)

[0326] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0327] ΔΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0328] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0329] Wherein, in the cell experiment, ACt(control group average) is the arithmetic mean of several ACt(control group) of the control group. In the animal experiment, ACt(control group average) is the arithmetic mean of the respective ACt(control group) of five mice of the control group at the same time point. Therefore, each sample of the test group and the control group corresponds to a ΔΔCt value.

[0330] Test group target gene mRNA relative expression level = 2 -ΔΔCt( Test group) x 100%

[0331] The test group target gene mRNA expression level is normalized based on the control group, and the control group target gene mRNA expression level is defined as 100%.

[0332] Test group target gene mRNA expression inhibition rate (%) = 1-test group target gene mRNA relative expression level

[0333] Unless otherwise specified, the in vivo activity experiment data are expressed as The experimental data are plotted and analyzed using GraphPad prism 8.0 software.

[0334] Example 1: Evaluation of the inhibitory activity of the 3' end NM054 modified sequence of the antisense strand on the target gene Superoxide dismutase 1 (SOD1) in mouse primary liver cells

[0335] Example 1: Using the above method for evaluating the inhibitory activity of target genes in mouse primary liver cells, the inhibitory activity of the antisense strand 20 NM054 modified sequence RZ599034 and the reference sequence RZ599001 on the target gene SOD1 in mouse primary liver cells was evaluated.

[0336] Mouse primary liver cells were seeded at a cell density of 2 x 10 5 The cells were seeded in 12-well culture plates at a cell density of 2 x 10

[0337] Table 5: Primer sequence information in Example 1

[0338] The results of Example 1 (Figure 1, Table 6) show that the antisense strand 20 position NM054 modified sequence RZ599034 has a stronger target gene inhibitory effect than the reference sequence RZ599001.

[0339] Table 6 Inhibitory activity of siRNA conjugates RZ599034 and reference sequence on target gene in mouse liver primary cells

[0340] Example 2. Evaluation of inhibitory activity of antisense strand 3' end NM054 modified sequence on target gene SOD1 in mice

[0341] This example evaluates the inhibitory activity of conjugate RZ599060 containing an NM054 group at the 20 position of the antisense strand of the SOD1 target siRNA, and conjugates RZ599061 containing an NM054 group at the 20 and 21 positions, and conjugate RZ599001 not containing the group, on the target gene SOD1 in mice using the method for evaluating target gene inhibitory activity in mice.

[0342] Six to eight week old C57BL / 6j mice were randomly divided into four groups by weight, 15 mice in each group, and each group of mice was administered the above siRNA conjugates by subcutaneous injection in the abdomen, wherein each mouse in the PBS control group was administered a dose of 5 mL / kg of the administration volume, and each mouse in the siRNA conjugate test group was administered a dose of 3 mg / kg (calculated as siRNA) of the administration volume of 5 mL / kg. The day of administration was designated as day 0 (D0), and 5 mice from each group were sacrificed on day 7 (D7), day 28 (D28), and day 49 (D49) after administration, and the animals were subjected to gross dissection, and the liver tissue was collected and cut into several 2 mm 3 The RNA extraction, reverse transcription reaction, and fluorescence quantitative PCR operation were performed as described above, and the gene expression difference was calculated by the ΔΔCt method. The primers are shown in Table N of Example 1.

[0343] The results of Example 2 (Figure 2, Table 7) show that conjugates RZ599060 and RZ599061 containing the NM054 group have higher inhibitory activity on D7, D28, and D49 than the control conjugate RZ599001 not containing the NM054 group, and the inhibitory activity of RZ599060 and RZ599061 is about 15% higher than that of the reference sequence RZ599001 on D49.

[0344] Table 7 Inhibitory activity of siRNA conjugates RZ599060 and RZ599061 on target gene in mice

[0345] Example 3. Evaluation of the inhibitory activity of 3' terminal NM054 modified sequences of antisense strand on target gene angiopoietin-like 3 (ANGPTL3) in mice

[0346] This example used the method for evaluating the inhibitory activity of target genes in mice to evaluate the inhibitory activity of conjugate RZ597115 containing NM054 group at position 20 of the antisense strand of ANGPTL3 target site siRNA, and conjugate RZ597116 containing NM054 at positions 20 and 21, and conjugate RZ597114 not containing the group on the target gene ANGPTL3 in mice.

[0347] 6-8 week old C57BL / 6j mice were randomly divided into 4 groups according to body weight, 15 mice in each group, and each group of mice was given the above siRNA conjugate by subcutaneous administration in the abdomen. The dose for each mouse in the PBS control group was 5 mL / kg of the administration volume, and the dose for each mouse in the siRNA conjugate experimental group was 3 mg / kg (calculated as siRNA) and 5 mL / kg of the administration volume. The day of administration was recorded as D0, and 5 mice from each group were sacrificed at D7, D28, and D56 after administration, and the animals were subjected to gross dissection, and the liver tissue was collected and cut into several 2 mm 3 small pieces were stored in RNAlater. RNA extraction, reverse transcription reaction, and Real-time PCR detection were performed as described above, and the gene expression difference was calculated by the ΔΔCt method.

[0348] Table 8. Primer sequence information in Example 3

[0349] The results of Example 3 (Figure 3, Table 9) showed that conjugates RZ597115 and RZ597116 containing the NM054 group had higher inhibitory activity at D7, D28, and D56 compared to the control conjugate RZ597114 not containing the NM054 group. The inhibitory activity of conjugate RZ597115 containing the NM054 group at position 20 of the antisense strand was about 25% higher than that of control conjugate RZ597114 at D56, and the inhibitory activity of conjugate RZ597116 containing the NM054 group at positions 20 and 21 was about 35% higher than that of control conjugate RZ597114.

[0350] Table 9. Inhibitory activity of target genes in mice after administration of siRNA conjugates RZ597115 and RZ597116

[0351] Example 4. Evaluation of the inhibitory activity of PS modified sequences on target gene Complement C3 (C3) in mouse liver primary cells

[0352] Example 4 evaluated the inhibitory activities of antisense PS-modified stepwise sequences RZ502053-RZ502068 at positions 3-4 to 18-19 (counted from the 5' end) and sense PS-modified stepwise sequences RZ502069-RZ502084 at positions 3-4 to 18-19 (counted from the 5' end) on the target gene C3 in mouse primary hepatocytes, in comparison with the reference sequence RZ002001 having phosphodiester linkage at the corresponding positions. The mouse primary hepatocytes were isolated and cultured as described above. The mouse primary hepatocytes were obtained from fresh liver tissues of C56BL / 6j mice, and the cell amount was 2x10 5 The 12-well culture plates were seeded with cells at a density of 2x10

[0353] Table 10 Primer sequence information in Example 4

[0354] The results of Example 4 (Figures 4 and 5, Tables 11 and 12) showed that the antisense PS-modified stepwise sequences RZ502053-RZ502068 at positions 3-4 to 18-19 had comparable or better inhibitory activities on the target gene than the reference sequence RZ002001. Among them, the PS-modified sequence RZ502055 at positions 5-6, the PS-modified sequences RZ502059-RZ502062 at positions 9-13, and the PS-modified sequences RZ502065-RZ502067 at positions 15-18 exhibited better inhibitory activities on the target gene. Except for the PS-modified sequence RZ502075 at positions 9-10, the sense PS-modified stepwise sequences RZ502069-RZ502084 at positions 3-4 to 18-19 had comparable or better inhibitory activities on the target gene than the reference sequence RZ002001. Among them, the sequence with additional PS modification at the 3' end of the sense strand exhibited better inhibitory activity on the target gene.

[0355] Table 11 Inhibition activity of target genes in mouse primary hepatocytes after administration of siRNA conjugates RZ502053-RZ502068 and reference sequences

[0356] Table 12 Inhibition activity of target genes in mouse primary hepatocytes after administration of siRNA conjugates RZ502069-RZ502084 and reference sequences

[0357] Example 5. Evaluation of inhibition activity of PS modified sequences on target gene complement factor B (CFB) in mice in vivo

[0358] In this example, the inhibition activity of sequence RZ011307 with PS modification between 18, 19 of sense strand and between 10, 11 of antisense strand and reference sequence RZ011306 on target gene CFB in mice in vivo was evaluated by the method for evaluating target gene inhibition activity in mice in vivo.

[0359] Six to eight weeks old C57BL / 6j mice were randomly divided into three groups according to body weight, 15 mice in each group. Each group of mice was administered with the above siRNA conjugates by subcutaneous injection in the abdomen, wherein each mouse in the PBS control group was administered with a dose of 5 ml / kg of the administration volume, and each mouse in the siRNA conjugate experimental group was administered with a dose of 3 mg / kg (calculated as siRNA) of the administration volume of 5 ml / kg. The day of administration was recorded as D0, and five mice in each group were sacrificed at D7, D28, and D56 after administration, respectively, for gross dissection of the animals, collection of liver tissues, cutting into several 2 mm 3 The RNA extraction, reverse transcription reaction, and Real-time PCR detection method were as described above, and the relative quantification of the target gene mRNA in each test group was calculated according to the aforementioned ΔΔCt method.

[0360] Table 13 Primer sequence information in Example 5

[0361] The results of Example 5 (Figure 6, Table 14) showed that sequence RZ011307 with PS modification between 18, 19 of sense strand and between 10-11 of antisense strand had better target gene inhibition activity and longer pharmacodynamic effect time compared with reference sequence RZ011306.

[0362] Table 14 Inhibition activity of target genes in mice in vivo after administration of siRNA conjugate RZ011307 and reference sequence

[0363] Example 6. Inhibition activity of antisense strand 3' end containing NM054 and PS-modified sequences on SEAP protein in serum of BALB / c-SEAP model was evaluated

[0364] In this example, the relative expression of SEAP protein in serum of BALB / c-SEAP model mice was evaluated for the sequences RZ003075 and the reference sequence RZ003089, which contained NM054 at positions 20 and 21 of the antisense strand and increased PS between positions 18 and 19 of the sense strand and between positions 10 and 11 of the antisense strand.

[0365] The BALB / c-SEAP model was constructed by inserting the AGT transcript sequence into a transposon plasmid (Shanghai Langjing Biotechnology Co., Ltd.), and 1.6 mL of plasmid mixed injection solution (25 μg of transposon plasmid fused with AGT transcript, 25 μg of transposase Super PiggyBac Transposase plasmid) was injected into the mouse through the tail vein within 3-5 s by fluid dynamics, and a mouse model stably expressing the SEAP reporter gene was obtained after 2 weeks.

[0366] Animal grouping, drug administration and serum sample collection:

[0367] Female BALB / c-SEAP stable transgenic mice were grouped according to SEAP levels, with 5 mice in each group. Each test group was given a predetermined dose of drug and a normal saline control group was added. RZ003089 and RZ003075 sequences were administered by abdominal subcutaneous injection, and all mice were calculated for drug dosage according to body weight, with a drug volume of 10 mL / kg (mouse weight) and a drug dosage of 0.3 mg / kg (calculated as siRNA / mouse weight). The normal saline control group was given a normal saline solution without siRNA conjugate at a dose of 10 mL / kg (mouse weight). Serum was collected from mice in the normal saline group and RZ003089 and RZ003075 administration groups on D5 after administration. The Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP levels in the serum of all groups of mice.

[0368] The results of Example 6 (Figure 7, Table 15) showed that compared with the reference sequence RZ003089, the RZ003075 sequence with increased PS and NM054 modification had a higher inhibitory effect on SEAP in the serum of mice.

[0369] Table 15 Relative expression levels of SEAP in the serum of mice administered with siRNA conjugate RZ003075 and the reference sequence

[0370] Example 7. Evaluation of the inhibitory activity of the sequences with 3' end of antisense strand containing NM054 and increased PS modification in both sense and antisense strands on the SEAP protein in serum of BALB / c-SEAP

[0371] This example was used to evaluate the effect of the sequence RZ003079 with 20, 21 positions of antisense strand containing NM054 and increased PS between 18, 19 positions of sense strand and between 10, 11 positions of antisense strand on the relative expression of SEAP in serum compared with the reference sequence RZ003087 in a mouse model with SEAP reporter gene.

[0372] The BALB / c-SEAP model was constructed as shown in Example 6. Female BALB / c-SEAP stable transgenic mice were grouped according to the SEAP level, 5 mice per group. Each test group was given a predetermined dose of drug and a normal saline control group was added. The RZ003087 and RZ003079 sequences were administered by a single dose of abdominal subcutaneous injection. The drug dose was calculated according to the body weight of the mice, and the volume of the drug was 10 mL / kg (body weight of the mice). The drug dose was 3 mg / kg (calculated as siRNA / mouse body weight). The normal saline control group was given a normal saline solution without siRNA conjugate at a volume of 10 mL / kg (mouse body weight). The serum of the mice in the normal saline group and the RZ003087 and RZ003079 administration groups was collected before administration (pre-dose) and at D7, D14, D21, D28, and D35 after administration. The SEAP level in the serum of the mice was detected by Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP level in the serum of the mice in all groups.

[0373] The results of Example 7 (Figure 8, Table 16) showed that compared with the reference sequence RZ003087, the RZ003079 sequence with increased PS and NM054 modification had a higher inhibitory effect on the expression of SEAP protein in the serum of the mice at each time point, and showed a longer protein inhibition level.

[0374] Table 16. Relative expression level of SEAP in the serum of mice administered with a single dose of siRNA conjugate RZ003079 and the reference sequence

[0375] Example 8. Evaluation of the inhibitory activity of the sequences with 3' end of antisense strand containing NM054 and increased PS modification in both sense and antisense strands on the SEAP protein in serum of BALB / c-SEAP

[0376] The mouse model with SEAP reporter gene was used to evaluate the effect of conjugate RZ003080 with NM054 at 20, 21 of antisense strand and increased PS between 18, 19 of sense strand and between 10, 11 of antisense strand on the relative expression of SEAP in serum compared with reference sequence RZ003088.

[0377] The BALB / c-SEAP model was constructed as shown in Example 6. Female BALB / c-SEAP stable transgenic mice were grouped according to the level of SEAP, 5 mice per group. Each test group was given a predetermined dose of drug and a normal saline control group was added. RZ003088 and RZ003080 were administered by subcutaneous injection in the abdomen in a single dose. The drug dose was calculated according to the body weight of the mice, and the volume of the drug was 10 mL / kg (body weight of the mice). The drug dose was 3 mg / kg (calculated as siRNA per mouse weight). The normal saline control group was given normal saline solution without siRNA conjugate at a volume of 10 mL / kg (mouse weight). The serum of the mice in the normal saline group and the RZ003080 and RZ003088 administration groups was collected before administration (pre-dose) and at D7, D14, D21, D28 and D35 after administration. The SEAP level in the serum of the mice was detected by Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP level in the serum of the mice in all groups.

[0378] The results of Example 8 (Figure 9, Table 17) show that compared with the reference sequence RZ003088, the RZ003080 with increased PS and NM054 modification has equivalent or higher inhibition of the expression of SEAP protein in the serum of the mice at each time point, and shows a longer protein inhibition level.

[0379] Table 17 shows the relative expression level of SEAP in the serum of the mice administered with siRNA conjugate RZ003080 and the reference sequence

[0380] Example 9. Evaluation of the inhibition activity of the sequence with NM054 at the 3' end of the antisense strand and increased PS modification of the sense and antisense strands on the SEAP protein in the serum of BALB / c-SEAP

[0381] The mouse model with SEAP reporter gene was used to evaluate the effect of conjugate RZ003076 with NM054 at 20, 21 of antisense strand and increased PS between 18, 19 of sense strand and between 10, 11 of antisense strand on the relative expression of SEAP in serum compared with reference sequence RZ003090.

[0382] The BALB / c-SEAP model was constructed as described in Example 6. Female BALB / c-SEAP stable transgenic mice were grouped according to SEAP levels, 5 mice per group. Each test group was given a predetermined dose of drug and an increasing saline control group. RZ003076, RZ003090 sequence single dose was administered by abdominal subcutaneous injection, all mice were calculated drug dose according to body weight, the drug volume was 10 mL / kg (mouse weight), the drug dose was 3 mg / kg (calculated by siRNA / mouse weight). The saline control group was given saline solution without siRNA conjugate 10 mL / kg (mouse weight). The serum of mice in saline group and RZ003076, RZ003090 administration group was collected before administration (pre-dose) and D7, D14, D21, D28, D35 after administration. The serum SEAP level was detected by Phospha-Light TM The SEAP reporter gene detection system (Thermo, T1017) was used to detect the SEAP level in the serum of all groups of mice.

[0383] The results of Example 7 (Figure 10, Table 18) show that, compared with the reference sequence RZ003090, RZ003076 with increased PS and NM054 modification has equivalent or higher inhibition of SEAP protein expression in the serum of mice at each time point, and shows longer protein inhibition levels.

[0384] Table 18 Relative expression level of SEAP in the serum of mice administered with siRNA conjugate RZ003076 and reference sequence single dose

[0385] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present disclosure, and are not limiting; although the present disclosure has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial applicability

[0386] The present disclosure provides double-stranded oligonucleotides, conjugates and compositions and their uses to improve the affinity of double-stranded oligonucleotides to target genes and the inhibition efficiency of target genes.

Claims

1. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, the nucleotide sequence of the sense strand comprising 17-25 consecutive nucleotides in the mRNA of a target gene expression, and 17-23 nucleotides in the sense strand and the antisense strand are at least partially reverse-complementary to form a duplex region; wherein, there are at least 5 phosphorothioate linkages in the duplex region; there is at least one overhang in the double-stranded oligonucleotide, and the overhang is not located at the 5' end of the antisense strand; the overhang comprises at least one [2'-R1-2'-R2] modified nucleotide, R1 represents a substituent group replacing 2'-OH of the ribose ring in a substituted nucleotide, R2 represents a substituent group replacing 2'-H of the ribose ring in a substituted nucleotide, R1 is selected from halogen, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy, and R2 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy.

2. The double stranded oligonucleotide of claim 1, wherein the antisense strand contains at least 3 phosphorothioate internucleosidic linkages in the duplex region, and the sense strand contains at least 2 phosphorothioate internucleosidic linkages in the duplex region; optionally, in the direction from 5' end to 3' end, the antisense strand contains at least 3 phosphorothioate internucleosidic linkages in the duplex region, and the 3 phosphorothioate internucleosidic linkages are respectively located between the 1st nucleotide and the 2nd nucleotide, between the 2nd nucleotide and the 3rd nucleotide, and between the 10th nucleotide and the 11th nucleotide of the antisense strand.

3. The double stranded oligonucleotide of claim 1, wherein there is at least one phosphorothioate linkage in the internucleosidic linkage between the overhang and the duplex region in the double-stranded oligonucleotide, and the internucleosidic linkage between the nucleotides in the overhang; optionally, the internucleosidic linkage between the overhang and the duplex region in the double-stranded oligonucleotide, and the internucleosidic linkage between the nucleotides in the overhang are all selected from phosphorothioate linkages; optionally, the overhang consists of 2-5 [2'-R1-2'-R2] modified nucleotides, and the two adjacent [2'-R1-2'-R2] modified nucleotides are connected by a phosphorothioate linkage; R1 is selected from F or methoxy, and R2 is selected from methyl; optionally, there is one overhang in the double-stranded oligonucleotide, and the overhang is located at the 3' end of the antisense strand.

4. The double-stranded oligonucleotide according to any one of claims 1 to 3, characterized in that, The double-stranded oligonucleotide has a structure shown in Formula (I): wherein SS represents the sense strand, and AS represents the antisense strand; o represents a phosphodiester internucleosidic linkage, and s represents a phosphorothioate internucleosidic linkage; B1, B2, B3 are each independently selected from [2'-R1-2'-R2] modified nucleotides, a1, e1, and a2 are each independently selected from an integer between 0 and 3, and at least one of a1, e1, and a2 is not 0; when a1 is not 0, represents an overhang at the 5' end of the sense strand; in the case that e1 is not 0, represents an overhang at the 3' end of the sense strand; when a2 is other than 0, represents an overhang located at the 3' end of the antisense strand; N1, N1', N2, N2', N x and N x each independently represents an unmodified or modified nucleotide, d1 and d2 are each independently selected from an integer of 2-4, and the two adjacent N2 are connected by a phosphodiester linkage or a phosphorothioate linkage, and the two adjacent N2' are connected by a phosphorothioate linkage; c1 and c2 are each independently selected from integers of 12-19, and adjacent two N x are phosphorodiamidate linkage or phosphorothioamidate linkage, and adjacent two N x are phosphorodiamidate linkage or phosphorothioamidate linkage, and there is at least one phosphorothioate linkage in ; b1 and b2 are each independently selected from an integer of 2-4, and the two adjacent N1 are connected by a phosphorothioate linkage, and the two adjacent N1' are connected by a phosphodiester linkage or a phosphorothioate linkage.

5. The double-stranded oligonucleotide of claim 4, wherein, the 10th nucleotide and the 11th nucleotide of the antisense strand are connected by a phosphorothioate linkage, counted from the 5' end; and / or, between the nucleotide in the sense strand that base pairs with the 10th nucleotide of the antisense strand and the nucleotide in the sense strand that base pairs with the 11th nucleotide of the antisense strand is a phosphodiester linkage; Optionally, a1 is selected from 0; Optionally, e1 is selected from 0; Optionally, a2 is selected from an integer from 1 to 3; Optionally, d1 is selected from an integer from 2 to 3; Optionally, between any two adjacent N2 is a phosphorothioate linkage; Optionally, b1 is selected from 3; Optionally, b2 is selected from 2; Optionally, between any two adjacent N1' is a phosphodiester linkage; Optionally, d2 is selected from 3.

6. The double-stranded oligonucleotide of claim 4, wherein, The double-stranded oligonucleotide has a structure represented by Formula (II): wherein each o / s independently represents a phosphodiester internucleosidic linkage or a phosphorothioate internucleosidic linkage; j1 is selected from 7; j1' is selected from 6; j2 is selected from an integer from 5 to 7, j2' is selected from an integer from 6 to 8, and j2' = j2 + 1.

7. The double-stranded oligonucleotide according to any one of claims 4-6, wherein, N1, N1', N2, N2', N x and N x each independently selected from modified nucleotides; each of the modified nucleotides is independently selected from at least one of a 2'-halogen modified nucleotide, a 2'-deoxy modified nucleotide, a 2'-O-R3modified nucleotide, a 2'-O-(CH2) n R4modified nucleotide, a [2'-F-2'-F] modified nucleotide, or a nucleotide analog; wherein the two F in the [2'-F-2'-F] modified nucleotide are substituent groups that replace the 2'-OH and 2'-H of the ribose ring of the nucleotide, respectively; R3 is selected from optionally substituted C1-C6 alkyl; n is selected from an integer from 0 to 3; R4is selected from an optionally substituted C1-C6alkoxy or R 4a , R 4b , and R 4c are each independently selected from optionally substituted C1-C6alkyl or optionally substituted C1-C6alkoxy; the nucleotide analogs comprise one or more of PNA, MNA, BNA, LNA, GNA, TNA, or UNA; Optionally, the 2'-halogen modified nucleotide is selected from a 2'-F modified nucleotide; Optionally, the 2'-O-R3 modified nucleotide is selected from a 2'-O-CH3 modified nucleotide; optionally, the 2'-0-(CH2) n -R4modified nucleotides are selected from 2'-0-TBDMS modified nucleotides, 2'-0-TIPS modified nucleotides, 2'-0-TOM modified nucleotides, 2'-0-CH2-0-CH2-CH3modified nucleotides, 2'-0-CH2-0-CH2-CF3modified nucleotides, or 2'-0-CH2-CH2-0-CH3modified nucleotides; optionally, the 2'-0-(CH2) n -R4modified nucleotides are selected from 2'-0-CH2-CH2-O-CH3.

8. The double-stranded oligonucleotide of claim 7, wherein, at least one 2'-0-(CH2) n -R4modified nucleotides; each of the 2'-0-(CH2) n -R4modified nucleotides are independently located at positions 5, 7, 8, 10, 12, 18, 19 of the sense strand or positions 8, 10, 15, 20, 21 of the antisense strand, counting from the 5' terminus. optionally, the double-stranded oligonucleotide contains one 2'-0-(CH2) n -R4 modified nucleotide, and the 2'-0-(CH2) n -R4 modified nucleotide is located on the antisense strand; optionally, the double-stranded oligonucleotide contains one 2'-0-(CH2) n -R4 modified nucleotide, and the 2'-0-(CH2) n -R4 modified nucleotide is located at position 15 from the 5' terminus of the antisense strand.

9. The double-stranded oligonucleotide of claim 7, wherein, the duplex region of the double stranded oligonucleotide comprises at least one [2'-F-2'-F] modified nucleotide; Optionally, the duplex region of the double stranded oligonucleotide comprises at least one [2'-F-2'-F] modified nucleotide; and at least one of the 2nd, 6th, 9th, 12th, 14th, 16th nucleotide of the antisense strand and the nucleotide in the sense strand that base pairs with the 10th, 11th, 12th, 13th, 15th nucleotide of the antisense strand is selected from a [2'-F-2'-F] modified nucleotide, counting from the 5' end; Optionally, the duplex region of the double stranded oligonucleotide comprises one [2'-F-2'-F] modified nucleotide, and the [2'-F-2'-F] modified nucleotide is located at the 14th position of the antisense strand, counting from the 5' end.

10. The double-stranded oligonucleotide of claim 7, wherein, the sense strand has 19 nucleotides, and the antisense strand has 21 nucleotides; at least four of the 2nd, 6th, 9th, 12th, 14th, 16th nucleotide of the antisense strand, counting from the 5' end to the 3' end, is selected from a 2'-F modified nucleotide, the 20th-21st nucleotide is selected from a [2'-F-2'-CH3] or [2'-CH3-2'-O-CH3] modified nucleotide, and the rest of the nucleotides are selected from 2'-O-CH3 modified nucleotides; and / or, at least three of the 7th-10th nucleotide of the sense strand, counting from the 5' end to the 3' end, is selected from a 2'-F modified nucleotide, and the rest of the nucleotides are selected from 2'-O-CH3 modified nucleotides.

11. The double-stranded oligonucleotide according to any one of claims 1 to 10, wherein the double stranded oligonucleotide is selected from an siRNA.

12. A conjugate, characterized in that, the conjugate comprises the double stranded oligonucleotide of any one of claims 1-11 and one or more targeted delivery ligands; Optionally, the targeted delivery ligand is selected from a liver-targeted delivery ligand, a kidney-targeted delivery ligand, a lung-targeted delivery ligand, a muscle-targeted delivery ligand, a fat-targeted delivery ligand, a brain-targeted delivery ligand, a heart-targeted delivery ligand, or a CNS-targeted delivery ligand.

13. A composition characterized in that, The composition comprises the conjugate according to claim 12.

14. Use of a double-stranded oligonucleotide according to any one of claims 1 to 11, and / or of a conjugate according to claim 12 and / or of a composition according to claim 13 for the manufacture of a medicament for the prevention and / or treatment of a disease.

15. Use of a double-stranded oligonucleotide according to any one of claims 1 to 11, and / or of a conjugate according to claim 12 and / or of a composition according to claim 13 for the manufacture of a medicament for reducing the expression or activity of a target gene.

16. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises a double-stranded oligonucleotide according to any one of claims 1 to 11, and / or a conjugate according to claim 12 and / or a composition according to claim 13 and a pharmaceutically acceptable excipient.

17. A method of reducing the expression or activity of a target gene, comprising contacting the target gene with a compound of claim 1. 0 The method comprises contacting a cell with a double-stranded oligonucleotide according to any one of claims 1 to 11, and / or a conjugate according to claim 12, and / or a composition according to claim 13 or a pharmaceutical composition according to claim 16.

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