Double-stranded oligonucleotides targeting INHBE gene, conjugates, compositions and uses thereof

Abstract

The invention provides double-stranded oligonucleotide targeting INHBE gene, a conjugate and a composition, and relates to the technical field of nucleic acid drugs. The double-stranded oligonucleotide comprises a positive-sense strand and an antisense strand, and the antisense strand and the positive-sense strand are complementary or basically complementary; the positive-sense strand comprises a nucleotide sequence identical or substantially identical to at least 15 contiguous nucleotides in the SEQ ID NO: 309 sequence, the substantially identical means that the positive-sense strand has no more than 3 nucleotide differences from the at least 15 contiguous nucleotides in the SEQ ID NO: 309 sequence. The double-stranded oligonucleotide can effectively inhibit the INHBE gene and treat patients suffering from metabolic disorders or metabolic syndromes and related diseases such as diabetes, hypertension and cardiovascular diseases.

Description

Double-stranded oligonucleotides, conjugates, compositions and uses thereof targeting INHBE gene

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311053101.0 filed with the State Intellectual Property Office of China on August 21, 2023, entitled “Double-stranded oligonucleotides, conjugates, compositions and uses thereof targeting INHBE gene,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the field of nucleic acid drug technology, and in particular relates to a double-stranded oligonucleotide targeting the INHBE gene, the double-stranded oligonucleotide conjugate, a pharmaceutical composition comprising the double-stranded oligonucleotide and / or the double-stranded oligonucleotide conjugate, and applications thereof. Background Art

[0004] Metabolic disorders are currently common, and typical drug treatments include lipid-lowering agents such as statins and other medications. However, these medications are often limited by frequent dosing and drug-drug interactions. Effective agents that can silence metabolic-related genes, such as those that inhibit INHBE, and thus effectively suppress metabolic disorders, are not yet commercially available.

[0005] Therefore, there is an urgent need in the art for drugs that can effectively inhibit the target INHBE gene, so as to treat subjects with metabolic disorders or metabolic syndrome and related diseases such as diabetes, hypertension and cardiovascular disease.

[0006] Summary of the Invention

[0007] In view of this, the present disclosure provides double-stranded oligonucleotides, conjugates, compositions, and uses thereof that target the INHBE gene. The present disclosure inhibits the expression of the INHBE gene by administering an INHBE-specific inhibitor (double-stranded oligonucleotide, conjugate, composition, etc.) to a subject to treat a disease or condition associated with the INHBE gene.

[0008] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0009] In a first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide targeting the INHBE gene, characterized in that the double-stranded oligonucleotide comprises a sense strand and an antisense strand, and the antisense strand is complementary or substantially complementary to the sense strand; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides.

[0010] Furthermore, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, the antisense strand comprising at least 15 consecutive nucleotides of any one of the sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides.

[0011] The sense strand comprises a nucleotide sequence that is at least partially reverse complementary or substantially complementary to the antisense strand to form a double-stranded region; the substantial complementarity means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides.

[0012] In some alternative embodiments of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0013] In a second aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide conjugate, wherein the conjugate comprises the double-stranded oligonucleotide and one or more ligands capable of binding to a cell receptor.

[0014] In some optional embodiments of the present disclosure, the ligand is conjugated to the sense strand and / or the antisense strand.

[0015] In a third aspect of the present disclosure, the present disclosure provides a composition comprising any one of the following:

[0016] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0017] (II) The conjugate according to the second aspect.

[0018] In a fourth aspect of the present disclosure, the present disclosure provides a use of any one of the following in the preparation of a medicament for preventing and / or treating a disease or condition mediated by the INHBE gene:

[0019] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0020] (II) the conjugate according to the second aspect; and / or

[0021] (III) The composition according to the third aspect.

[0022] In some optional embodiments of the present disclosure, the disease or condition includes, but is not limited to, having or being at risk for developing a metabolic disorder, type 2 diabetes, obesity, elevated triglyceride levels, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes, non-alcoholic steatohepatitis (NASH), cardiovascular disease, cardiomyopathy, hypertension, and / or heart failure.

[0023] In a fifth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any one of the following and a pharmaceutically acceptable excipient or adjuvant:

[0024] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0025] (II) the conjugate according to the second aspect; and / or

[0026] (III) The composition according to the third aspect.

[0027] In a sixth aspect of the present disclosure, the present disclosure provides a method for reducing the expression or activity of the INHBE gene, comprising contacting any one of the following with a cell:

[0028] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0029] (II) the conjugate according to the second aspect; and / or

[0030] (III) the composition of the third aspect; and / or

[0031] (IV) The pharmaceutical composition described in the fifth aspect.

[0032] In a seventh aspect of the present disclosure, the present disclosure provides a method for preventing and / or treating a disease or condition mediated by the INHBE gene, comprising administering to a subject a pharmaceutically acceptable amount of any one of the following:

[0033] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0034] (II) the conjugate according to the second aspect; and / or

[0035] (III) the composition of the third aspect; and / or

[0036] (IV) The pharmaceutical composition described in the fifth aspect.

[0037] Those skilled in the art will appreciate that modified nucleotide groups can be introduced into the double-stranded oligonucleotide conjugates of the present invention by using correspondingly modified nucleoside monomers. Methods for preparing correspondingly modified nucleoside monomers and methods for introducing modified nucleotide groups into double-stranded oligonucleotide conjugates are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1. Inhibitory activity of target genes in Balb / c-HDI mice after administration of siRNA conjugates.

[0039] Figure 2. Inhibitory activity of target genes in C57BL / 6J mice after administration of siRNA conjugates.

[0040] Figure 3 Inhibitory activity of target genes in C57BL / 6J mice after administration of different doses of RZM08019.

[0041] Fig. 4 Serum triglyceride levels in BKS-DB mice after repeated administration of RZM08019.

[0042] Fig. 5 Total cholesterol levels in serum of BKS-DB mice after repeated administration of RZM08019.

[0043] Figure 6. Inhibitory activity of target genes in Huh7 cells after administration of siRNA conjugates. DETAILED DESCRIPTION

[0044] The following is a clear and complete description of the technical solutions in the embodiments of the present disclosure. Obviously, the embodiments described are only some of the embodiments of the present disclosure, not all of them. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present disclosure.

[0045] Explanation of terms

[0046] Unless otherwise indicated, the following definitions used herein shall apply. For the purposes of this disclosure, chemical elements are consistent with the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry can be found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference. Alkylene can be substituted or unsubstituted.

[0047] As used herein, "halogen" or "halo" refers to any of the radioactive-stable atoms in column 7 of the periodic table, such as fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred, and fluorine being more preferred.

[0048] "Alkyl" as used in the present disclosure refers to a straight or branched alkane chain that is fully saturated (i.e., does not contain double or triple bonds). Alkyl can have 1 to 6 carbon atoms (whenever it appears in the text, a numerical range such as "1 to 6" refers to each integer in a given range; for example, "1 to 6 carbon atoms" means that the alkyl can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms, but this definition also encompasses the term "alkyl" in which no numerical range is specified). As an example only, "C1-C4 alkyl" means that there are one to four carbon atoms in the alkyl chain, i.e., the C1-C4 alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Typical alkyl groups include but are limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, etc. Alkyl can be substituted or unsubstituted.

[0049] As used herein, "alkoxy" refers to the formula -OR, wherein R is an alkyl group as defined above, for example, "C1-6 alkoxy", which includes but is not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy and tert-butoxy, etc.

[0050] As used herein, "alkylene" refers to a fully saturated, straight or branched alkylene chain of formula -R-. By way of example only, "C1-10 alkylene" means a group having from one to ten carbon atoms in the alkyl chain, i.e., the C1-10 alkylene group is selected from methylene (-CH2-), ethylene (=CH2CH3), 1,2-ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), and the like.

[0051] As used herein, the articles "a," "an," and "the" are intended to include "at least one" or "one or more." Thus, as used herein, these articles refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., more than one component may be contemplated for use or use in implementing the described embodiments.

[0052] In this disclosure, the terms "include," "comprising," "having," "may," "containing," and variations thereof are generally intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "consisting of" generally indicates that no other components (or, similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, nouns with an indefinite number also include plural referents.

[0053] In this disclosure, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0054] The term “comprising” used in the present disclosure is an open expression, which includes the contents specified in the present disclosure but does not exclude other contents.

[0055] As used in this disclosure, "optionally substituted" is used to define a variable, which may be unsubstituted.

[0056] As used in this disclosure, "unsubstituted" means that the designated group bears no substituents.

[0057] As used herein, the terms "substituted," "substituted," and "substituted" are used interchangeably to indicate that one or more hydrogen atoms in a given structure are replaced with a specified substituent. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structure is substitutable with one or more substituents selected from a specified group, the substituents may be the same or different at each substitutable position.

[0058] As used in this disclosure, “each... is independently selected from” and “... are each independently selected from” and “... are independently selected from” are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0059] In this disclosure, the terms "include," "comprising," "having," "may," "containing," and variations thereof are generally intended to be open transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "consisting of" generally indicates that no other components (or, similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, nouns with an indefinite number also include plural referents.

[0060] As used herein, "small interfering RNA (siRNA)" is a type of double-stranded RNA comprising a sense strand and an antisense strand. siRNAs mediate the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway by forming a silencing complex. Specifically, siRNAs direct the specific degradation of mRNA sequences through the well-known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and protein.

[0061] In the present disclosure, a double-stranded oligonucleotide is composed of two chains, wherein the chain that binds to the target sequence is called the antisense chain or guide chain, and the other chain is called the sense chain or passenger chain. The term "antisense chain" refers to a chain of a double-stranded oligonucleotide that includes a region that is completely or substantially complementary to the target sequence. The term "sense chain" refers to a chain of a double-stranded oligonucleotide that includes a region that is substantially complementary to the region of the antisense chain as defined herein. The term "complementary region" refers to a region on the antisense chain that is completely or substantially complementary to the target sequence. In the case where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the interior or terminal regions of the molecule. As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. Herein, double-stranded oligonucleotides and siRNA can be used interchangeably.

[0062] In the present disclosure, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases on the other strand in a complementary manner.

[0063] In the present disclosure, unless otherwise specified, "substantially reverse complementary" or "substantially complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences.

[0064] In the present disclosure, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleotide sequences are considered to have a nucleotide difference at that position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleotide difference at that position can also be considered.

[0065] In the present disclosure, "fluorinated nucleotides" or "2'-fluorinated modified nucleotides" refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and "non-fluorinated modified nucleotides" refer to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. The "methoxy-modified nucleotides" or "2'-O-methoxyethyl-modified nucleotides" refer to nucleotides in which the 2' hydroxyl group of the ribose group is replaced by a methoxy group or a methoxyethyl group. "Methoxy-modified nucleotides" are also described as 2'-OMe or 2'-O-methyl-modified nucleotides, which can be used interchangeably. "2'-O-methoxyethyl-modified nucleotides" are also described as 2'-MOE modifications. 2'-deoxynucleotides refer to nucleotides in which the 2' position of the ribose group is hydrogen.

[0066] The terms "RNAi", "iRNA", "RNAi agent", "RNAi reagent", "RNA interference agent", and "RNA inhibitor" are used interchangeably herein to refer to molecules or agents that contain RNA and can mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. It is well known in the art that RNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). In one embodiment, the RNAi agents of the present disclosure include single-stranded or double-stranded RNA that interacts with a target RNA sequence to guide the cleavage of the target RNA. Therefore, in one aspect, the present disclosure relates to the term "siRNA" which can also be used to refer to the RNAi described above.

[0067] In some embodiments, the "RNAi" used in the compositions, uses, and methods of the present disclosure is double-stranded RNA, and the "RNAi agent" includes the double-stranded RNA, and the "RNAi agent" can refer to a "double-stranded RNAi agent", a "double-stranded RNA (dsRNA) molecule", a "dsRNA agent", a "siRNA agent", or a "dsRNA agent".

[0068] As used herein, the term "dsRNA" or "siRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as "sense" and "antisense" orientations relative to the target RNA.

[0069] In addition, as used in this specification, "RNAi reagents" or "RNAi agents" can include ribonucleotides with chemical modifications and / or ligands; RNAi reagents can include substantial modifications at multiple nucleotides. The term "modified nucleotides" refers to nucleotides that independently have modified sugar moieties, modified internucleotide linkages, and / or modified nucleobases. Thus, the term modified nucleotides encompasses substitutions, additions, or removals of, for example, functional groups or atoms of internucleotide linkages, sugars, or nucleobases. Modifications suitable for use in the agents of the present disclosure include all types of modifications disclosed herein or known in the art. As used in siRNA molecules, any such modification can be encompassed by "RNAi agent."

[0070] In the present disclosure, the term "nucleotide overhang" or "overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA (e.g., dsRNA). For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. The one or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0071] In this disclosure, the term "ligand" generally refers to any compound or molecule that can be covalently or otherwise chemically bound to a biologically active substance (such as an oligonucleotide). In certain embodiments, a ligand can interact directly or indirectly with another compound, such as a receptor. The receptor that interacts with the ligand can be present on the cell surface, or alternatively can be an intracellular and / or intercellular receptor. The interaction of the ligand with the receptor can result in a biochemical reaction, or can be simply a physical interaction or binding.

[0072] The term "linked," when referring to a connection between two molecules, means that the two molecules are joined by a covalent bond or that the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond).

[0073] Each nucleotide in the sense strand and the antisense strand is independently a modified or unmodified nucleotide. In the context of the present invention, unless otherwise indicated, "conjugation" refers to the covalent bonding of two or more chemical moieties each having a specific function to each other; accordingly, "conjugate" refers to a compound formed by covalent bonding between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalent bonding of one or more chemical moieties having a specific function to siRNA. Hereinafter, the siRNA conjugate of the present invention will sometimes be referred to as a "conjugate". The siRNA conjugate should be understood as a general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense chain conjugate or the siRNA antisense chain conjugate, depending on the context.

[0074] In this disclosure, the term "pharmaceutical composition" or "composition" may refer to a composition for use in treating a disease or in vitro cell culture experiments. When used in treating a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more auxiliary ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0075] In this disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" in this disclosure means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0076] As used herein, "pharmaceutically acceptable carriers or excipients" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific intended dosage form. Except to the extent that any conventional excipient is incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0077] As used herein, the terms "treat," "treat," "alleviate," or "amortize" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. As used herein, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0078] As used herein, "prevention" and "prevent" are used interchangeably to refer to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0079] In the present disclosure, the term "administer" generally refers to introducing the disclosed pharmaceutical formulation into the body of a subject by any introduction or delivery route. Any method known to those skilled in the art for contacting cells, organs or tissues with the drug can be used. The administration may include, but is not limited to, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous or oral administration. The daily dose may be divided into one, two or more doses in a suitable form for administration at one, two or more times during a certain time period.

[0080] In the present disclosure, the term "contact" generally refers to the contacting of two or more different types of substances in any order, in any manner, and for any duration. Contact can occur in vivo, ex vivo, or in vitro. In some embodiments, it can refer to direct contact of a RNAi agent or composition of the present disclosure with a cell or tissue. In other embodiments, the term refers to indirect contact of a RNAi agent or composition of the present disclosure with a cell or tissue.

[0081] In this disclosure, the term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, improvement, prevention and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adult subjects. Subjects include animal disease models.

[0082] In the present disclosure, the term "modulate gene expression" means that the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits is upregulated or downregulated so that the expression, level or activity is greater or less than that observed in the absence of the modulator. For example, the term "modulate" may mean "inhibit," but the use of the word "modulate" is not limited to this definition.

[0083] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0084] Double-stranded oligonucleotide targeting the INHBE gene

[0085] In a first aspect, the present disclosure provides a double-stranded oligonucleotide targeting the INHBE gene, which is capable of inhibiting INHBE gene expression in mammals, including humans, monkeys, rats, or mice. The double-stranded oligonucleotide is capable of inhibiting INHBE gene expression in cells in vitro and in vivo.

[0086] Specifically, the present disclosure provides a double-stranded oligonucleotide targeting the INHBE gene, characterized in that the double-stranded oligonucleotide includes a sense chain and an antisense chain, and the antisense chain is complementary or substantially complementary to the sense chain; the substantially complementary means that the mismatch between the sense chain and the antisense chain in the double-stranded region does not exceed 3 nucleotides.

[0087] The positive strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the SEQ ID NO: 309 sequence, and the substantially identical sequence means that the positive strand has no more than 3 nucleotide differences from the at least 15 consecutive nucleotides in the SEQ ID NO: 309 sequence.

[0088] In an optional embodiment of the present disclosure, the sense strand comprises a nucleotide sequence that differs from at least 15 consecutive nucleotides in the sequence of SEQ ID NO: 309 by no more than 2 nucleotides, preferably by no more than 1 nucleotide.

[0089] In an optional embodiment of the present disclosure, the antisense strand is complementary or substantially complementary to at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 309, and the substantially complementary refers to that the mismatch in the complementary region does not exceed 3 nucleotides.

[0090] In an optional embodiment of the present disclosure, the double-stranded oligonucleotide includes a sense strand and an antisense strand, and the antisense strand comprises at least 15 consecutive nucleotides of any one of the sequences shown in SEQ ID NO:155 to SEQ ID NO:308 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides.

[0091] The sense strand comprises a nucleotide sequence that is at least partially reverse complementary or substantially complementary to the antisense strand to form a double-stranded region; the substantial complementarity means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides.

[0092] In some optional embodiments of the present disclosure, the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any one of the sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 3 nucleotides.

[0093] In some optional embodiments of the present disclosure, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any one of the sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 2 nucleotides.

[0094] In some optional embodiments of the present disclosure, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any one of the sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 1 nucleotide.

[0095] In some optional embodiments of the present disclosure, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NO.155 to SEQ ID NO.308 in Table 1.

[0096] In some specific embodiments of the present disclosure, the antisense strand is selected from or comprises any one of the nucleotide sequences shown in SEQ ID NO.155 to SEQ ID NO.308 in Table 1.

[0097] In an optional embodiment of the present disclosure, as measured in the 5'-3' direction, positions 2-19 of the antisense strand comprise at least 15 nucleotides of nucleotides 2-19 of any one of the nucleotide sequences shown in SEQ ID NO.155 to SEQ ID NO.308 in Table 1, or a nucleotide sequence that differs from the at least 15 nucleotides by no more than 3 nucleotides.

[0098] In some optional embodiments of the present disclosure, the positive strand comprises at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 3 nucleotides.

[0099] In some optional embodiments of the present disclosure, the positive strand comprises at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 2 nucleotides.

[0100] In some optional embodiments of the present disclosure, the positive strand comprises at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 1 nucleotide.

[0101] In some optional embodiments of the present disclosure, the positive strand comprises at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1.

[0102] In some specific embodiments of the present disclosure, the sense strand is selected from or comprises any one of the nucleotide sequences shown in SEQ ID NO. 1 to SEQ ID NO. 154 in Table 1.

[0103] In some optional embodiments of the present disclosure, the double-stranded oligonucleotide comprises one or more of the duplex groups shown in the following numbers in Table 1: RN008015, RN008125, RN008128, RN008142, RN008145, RN008148.

[0104] In some alternative embodiments of the present disclosure, each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0105] The structural formula of nucleotides is Wherein, Base represents a nucleoside base, and the nucleoside base on each nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

[0106] In some optional embodiments of the present disclosure, substantially all of the nucleotides in the sense strand or the antisense strand are selected from modified nucleotides. Wherein, “substantially all of the nucleotides in the sense strand are selected from modified nucleotides” means that most but not all of the nucleotides in the sense strand are modified nucleotides, and may contain no more than 5, 4, 3, 2 or 1 unmodified nucleotides. “Substantially all of the nucleotides in the antisense strand are selected from modified nucleotides” means that most but not all of the nucleotides in the antisense strand are modified nucleotides, and may contain no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0107] In some specific embodiments of the present disclosure, all nucleotides of the sense strand or the antisense strand are selected from modified nucleotides.

[0108] In some optional embodiments of the present disclosure, the modified nucleotides are each independently selected from 2'-halo, 2'-deoxy, 2'-O-(CH2) n -R1 modified nucleotides, or quasi-nucleotides; the quasi-nucleotides are selected from one or more of peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA), and unlocked nucleic acid (UNA);

[0109] n is selected from 0, 1 or 2; R1 is selected from optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkoxy or -Si(R 1a )3; Each R 1a Independently selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy.

[0110] In the present disclosure, a 2'-halogenated modified nucleotide is one in which the 2'-hydroxyl group is replaced by a halogen atom. For example, a 2'-fluoro modified nucleotide has the structural formula

[0111] In the present disclosure, a 2'-deoxy modified nucleotide is a nucleotide in which the hydroxyl group at the 2' position is replaced by a hydrogen atom. Its structural formula is

[0112] In the present disclosure, 2'-O-(CH2) n-R1 modified nucleotides are nucleotides in which the hydrogen atom on the 2' hydroxyl group is replaced by -(CH2) n -R1 substituted, wherein 2'-O-(CH2) n The structural formula of the -R1 modified nucleotide is For example: when n is selected from 0, 2'-O-(CH2) n -R1 can be selected from 2'-O-methyl (also known as 2'-methoxy), 2'-O-methoxymethyl, 2'-O-TBDMS, 2'-O-TIPS or 2'-O-TOM. When n is selected from 1, 2'-O-(CH2) n -R1 can be selected from 2'-O-CH2-O-CH2-CH3 (2'-O-ethoxymethyl) or 2'-O-CH2-O-CH2-CF3 (also known as 2'-O-(2,2,2-trifluoroethoxymethyl)). When n is selected from 2, 2'-O-(CH2) n -R1 can be selected from 2'-O-CH2-CH2-O-CH3 (also known as 2'-O-methoxyethyl, 2'-O-MOE). Wherein, the structural formula of TBDMS is The structural formula of TIPS is The structural formula of TOM is

[0113] In some alternative embodiments of the present disclosure, 2'-O-(CH2) n -R1 is selected from 2'-O-CH3, 2'-O-CH2-O-CH3, 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3 or 2'-O-CH2-CH2-O-CH3.

[0114] In some optional embodiments of the present disclosure, the sense strand or the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0115] In some alternative embodiments of the present disclosure, the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0116] In some optional embodiments of the present disclosure, the sense strand or the antisense strand comprises a 3' overhang of at least 2 nucleotides.

[0117] In some alternative embodiments of the present disclosure, the antisense strand comprises a 3' overhang of at least 2 nucleotides.

[0118] In some specific embodiments of the present disclosure, the antisense strand comprises a 3' overhang of 2 nucleotides.

[0119] In some alternative embodiments of the present disclosure, the sense strand and / or the antisense strand independently comprise one or more phosphorothioate bonds.

[0120] In some specific embodiments of the present disclosure, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end.

[0121] In some specific embodiments of the present disclosure, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

[0122] In some optional embodiments of the present disclosure, all nucleotides of the sense strand and all nucleotides of the antisense strand are selected from modified nucleotides; wherein the double-stranded region formed by the sense strand and the antisense strand is represented by the following formula (I):

[0123] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0124] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(I),

[0125] Among them, SS represents the sense strand and AS represents the antisense strand;

[0126] Each N is independently selected from a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-deoxy-modified nucleotide;

[0127] Each X is independently selected from 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3, 2'-O-CH2-CH2-O-CH3;

[0128] Said a, a', p, p', b, b', q, q', c, c', r', and d' each independently represent the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0 at the same time, and 0≤q'+r'≤4.

[0129] In some optional embodiments of the present disclosure, a' is selected from an integer of 3-8, p' is selected from 0 or 1, b' is selected from an integer of 4-13, q' is selected from 0 or 1, and c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, p is selected from 1, b is selected from an integer of 4-8, q is selected from 0 or 1, and c is selected from an integer of 6-10.

[0130] In some alternative embodiments of the present disclosure, each N is independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides.

[0131] In some optional embodiments of the present disclosure, each X is independently selected from 2'-O-methyl modified nucleotides, 2'-O-MOE (methoxyethyl) modified nucleotides, 2'-O-TBDMS modified nucleotides, 2'-O-TIPS modified nucleotides, 2'-O-TOM modified nucleotides, 2'-O-CH2-O-CH2-CH3 modified nucleotides, 2'-O-CH2-O-CH2-CF3 modified nucleotides.

[0132] Preferably, each X is independently selected from 2'-O-methyl modified nucleotides, 2'-O-MOE modified nucleotides, 2'-O-CH2-O-CH2-CH3 modified nucleotides, 2'-O-CH2-O-CH2-CF3 modified nucleotides;

[0133] In some alternative embodiments of the present disclosure, the double-stranded region comprises at least one 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide.

[0134] In some alternative embodiments of the present disclosure, the double-stranded region comprises at least one 2'-O-methoxyethyl modified nucleotide.

[0135] In some specific embodiments of the present disclosure, the double-stranded region comprises a 2'-O-methoxyethyl modified nucleotide.

[0136] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0137] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0138] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0139] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0140] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0141] In some optional embodiments of the present disclosure, the antisense strand contains at least one 2'-O-methoxyethyl-modified nucleotide or a 2'-O-ethoxymethyl-modified nucleotide.

[0142] In some optional embodiments of the present disclosure, the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

[0143] In some specific embodiments of the present disclosure, the antisense strand contains a 2'-O-methoxyethyl modified nucleotide.

[0144] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl-modified nucleotide, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0145] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl-modified nucleotide, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0146] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense chain is selected from a 2'-O-methoxyethyl-modified nucleotide, and any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0147] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl-modified nucleotide, the nucleotides at positions 2, 6, 9, 14, and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0148] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl-modified nucleotide, the nucleotides at positions 2, 6, 12, 14, and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0149] In some optional embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7 to 10 of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides;

[0150] In some specific embodiments of the present disclosure, in the direction from 5' end to 3' end, the nucleotides at positions 7-10 of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0151] In some optional embodiments of the present disclosure, the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any modified antisense strand nucleotide sequence shown in Table 2, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 3 nucleotides.

[0152] In some optional embodiments of the present disclosure, the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any modified antisense strand nucleotide sequence shown in Table 2, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 2 nucleotides.

[0153] In some optional embodiments of the present disclosure, the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any modified antisense strand nucleotide sequence shown in Table 2, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 1 nucleotide.

[0154] In some optional embodiments of the present disclosure, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any modified antisense strand nucleotide sequence shown in Table 2.

[0155] In some specific embodiments of the present disclosure, the antisense strand is selected from or comprises any one of the modified antisense strand nucleotide sequences shown in Table 2.

[0156] In some optional embodiments of the present disclosure, the sense chain comprises at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any modified sense chain nucleotide sequence shown in Table 2, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides.

[0157] In some specific embodiments of the present disclosure, the sense strand of the double-stranded oligonucleotide is selected from or comprises any modified sense strand nucleotide sequence shown in Table 2.

[0158] In some specific embodiments of the present disclosure, the sense strand of the double-stranded oligonucleotide is selected from or comprises any modified sense strand nucleotide sequence shown in Table 2; the antisense strand is selected from or comprises any modified antisense strand nucleotide sequence shown in Table 2.

[0159] In an optional embodiment of the present disclosure, the double-stranded oligonucleotide is selected from or comprises one or more of the modified duplex groups shown in the following numbers in Table 2: RX008157, RX008174, RX008177, RX008180, RX008182, and RX008185.

[0160] In some specific embodiments of the present disclosure, the double-stranded oligonucleotide is selected from siRNA.

[0161] Double-stranded oligonucleotide conjugates

[0162] In a second aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide conjugate, wherein the conjugate comprises the double-stranded oligonucleotide according to the first aspect and one or more ligands capable of binding to a cell receptor.

[0163] In some optional embodiments of the present disclosure, the ligand is conjugated to the sense strand and / or the antisense strand.

[0164] In some optional embodiments of the present disclosure, the ligand is conjugated to the 3' end and / or the 5' end of the sense strand.

[0165] In some specific embodiments of the present disclosure, the ligand is conjugated to the 3' end of the sense strand.

[0166] In some specific embodiments of the present disclosure, the cellular receptor is selected from asialoglycoprotein receptors.

[0167] In some embodiments of the present disclosure, the ligand is selected from a galactose cluster. The galactose cluster comprises molecules having 2-4 terminal galactose derivatives. As used in the present disclosure, the galactose derivative comprises galactose and / or a galactose derivative having an affinity for asialoglycoprotein receptors equal to or greater than that of galactose.

[0168] In some specific embodiments of the present disclosure, the galactose cluster comprises a molecule having 2-4 terminal N-acetylgalactosamines (GalNAc).

[0169] In some optional embodiments of the present disclosure, the ligand is selected from the structure shown in formula (101) or its isomers or pharmaceutically acceptable salts thereof:

[0170] Wherein, * represents the conjugation site of the ligand and the sense strand or the antisense strand;

[0171] m is selected from 1, 2, 3 or 4;

[0172] Each Z is independently selected from hydroxyl or thiol;

[0173] Each p is independently selected from 1, 2 or 3;

[0174] Each q is independently selected from 1, 2 or 3;

[0175] Each R is independently selected from H, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy;

[0176] Each L is independently selected from an optionally substituted C2-C20 alkylene or R La and RLa independently selected from optionally substituted C1-C10 alkylene, k is selected from 1, 2, 3, 4 or 5;

[0177] Each Y is independently selected from O, S or NH.

[0178] In some optional embodiments of the present disclosure, m is selected from 1, 2 or 3.

[0179] In some specific embodiments of the present disclosure, m is selected from 3.

[0180] In some specific embodiments of the present disclosure, Z is selected from hydroxyl.

[0181] In some specific embodiments of the present disclosure, p is selected from 1.

[0182] In some specific embodiments of the present disclosure, q is selected from 1.

[0183] In some specific embodiments of the present disclosure, R is selected from H.

[0184] In some optional embodiments of the present disclosure, each L is independently selected from C1-C10 alkylene or Among them, R La and R La are independently selected from C1-C5 alkylene, and k is 1, 2 or 3.

[0185] In some specific embodiments of the present disclosure, k is selected from 1;

[0186] In some optional embodiments of the present disclosure, each L is independently selected from

[0187] In some specific embodiments of the present disclosure, Y is selected from O.

[0188] In some optional embodiments of the present disclosure, the ligand includes at least one of the following structures or isomers thereof or pharmaceutically acceptable salts thereof:

[0189] In some specific embodiments of the present disclosure, the ligand is selected from the following structures or isomers or pharmaceutically acceptable salts thereof:

[0190] In some optional embodiments of the present disclosure, the double-stranded oligonucleotide conjugate is selected from one or more of the conjugates shown in the following numbers in Table 4: RZ08040, RZ08041, RZ08042, RZ08043, RZ08044, and RZ08045.

[0191] In some specific embodiments of the present disclosure, the ligand is selected from the structure shown in formula (201):

[0192] Wherein, * represents the conjugation site of the ligand and the sense chain or the antisense chain.

[0193] Composition

[0194] In a third aspect of the present disclosure, the present disclosure provides a composition comprising any one of the following:

[0195] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0196] (II) The conjugate according to the second aspect.

[0197] Uses in treating diseases

[0198] In a fourth aspect of the present disclosure, the present disclosure provides a use of any one of the following in the preparation of a medicament for preventing and / or treating a disease or condition mediated by the INHBE gene:

[0199] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0200] (II) the conjugate according to the second aspect; and / or

[0201] (III) The composition according to the third aspect.

[0202] In some optional embodiments of the present disclosure, the disease or condition includes, but is not limited to, having or being at risk for developing a metabolic disorder, type 2 diabetes, obesity, elevated triglyceride levels, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes, non-alcoholic steatohepatitis (NASH), cardiovascular disease, cardiomyopathy, hypertension, and / or heart failure.

[0203] Pharmaceutical composition

[0204] In a fifth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising any one of the following and a pharmaceutically acceptable excipient or adjuvant:

[0205] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0206] (II) the conjugate according to the second aspect; and / or

[0207] (III) The composition according to the third aspect.

[0208] Methods for reducing INHBE gene expression or activity in vitro

[0209] In a sixth aspect of the present disclosure, the present disclosure provides a method for reducing the expression or activity of the INHBE gene, comprising contacting any one of the following with a cell:

[0210] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0211] (II) the conjugate according to the second aspect; and / or

[0212] (III) the composition of the third aspect; and / or

[0213] (IV) The pharmaceutical composition described in the fifth aspect.

[0214] Treatments for the disease

[0215] In a seventh aspect of the present disclosure, the present disclosure provides a method for preventing and / or treating a disease or condition mediated by the INHBE gene, comprising administering to a subject a pharmaceutically acceptable amount of any one of the following:

[0216] (I) the double-stranded oligonucleotide of the first aspect; and / or

[0217] (II) the conjugate according to the second aspect; and / or

[0218] (III) the composition of the third aspect; and / or

[0219] (IV) The pharmaceutical composition described in the fifth aspect.

[0220] The mRNA sequence encoding the INHBE gene of the present invention (SEQ ID NO: 309, NM_031479.5) is as follows:

[0221] Unless otherwise stated, the ratios of reagents used in each example of the present disclosure are calculated on a volume basis (v / v).

[0222] Unless otherwise stated, the reagents used in the examples of the present disclosure were purchased from Beijing Coupling Technology Co., Ltd., and the information of the main reagents is shown below.

[0223] Preparation of ligands

[0224] Preparation Example 1 Preparation of Compound CR01008

[0225] (1.1) Synthesis of compound CR01008

[0226] The synthetic route of compound CR01008 is as follows:

[0227] (1.1.1) Synthesis of Compound 2

[0228] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3% by mass KOH aqueous solution was added dropwise. After the addition was complete, the mixture was stirred at 25 ° C for 30 minutes, heated to 60 ° C and refluxed at 60 ° C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry, and filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M + H] + .

[0229] (1.1.2) Synthesis of compound 3

[0230] Compound 2 (9 g, 1 eq) prepared in step (1.1.1) was dissolved in 70 mL of 1,4-dioxane. A 4 M solution of hydrogen chloride in 1,4-dioxane (45 mL) was added, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0231] (1.1.3) Synthesis of compound 5

[0232] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) prepared in step (1.1.2) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under a N2 atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (22% acetonitrile in water) to obtain compound 5 (1.78 g, 64.4% yield) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0233] (1.1.4) Synthesis of Compound 6

[0234] Compound 5 (1.54 g, 1.0 eq) prepared in step (1.1.3) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (60% acetonitrile in water) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0235] (1.1.5) Synthesis of compound CR01008

[0236] Compound 6 (1.08 g, 1.0 eq) prepared in step (1.1.4) was dissolved in 20 ml of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. After reverse purification (72 vol% acetonitrile in water), the mixture was dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0237] 1HNMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37–1.17(m,5H),1.60–1.40(m,6H),1.68–1.62(m,1H),1.80(s,3H) ,1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58 –3.39(m,3H),3.69–3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H ),5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93–6.87(m,4H),7.35–7.21(m,7H),7.44–7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0238] Preparation Example 2 Preparation of Compound CR01008Z

[0239] Compound CR01008Z was obtained by linking compound 6, which was used to synthesize compound CR01008, to a solid phase support CPG.

[0240] The synthetic route of compound CR01008Z is as follows:

[0241] (1.2.1) Synthesis of compound 9

[0242] Compound 6 (500 mg) prepared in step (1.1.4) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0243] (1.2.2) Synthesis of compound CR01008Z

[0244] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (1.2.1) were added to a 20 ml sample vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0245] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0246] Preparation Example 3 Preparation of Compound CR01013

[0247] The synthetic route of compound CR01013 is as follows:

[0248] (1.3.1) Synthesis of Compound 2

[0249] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 4.9 g) was dissolved in 17 ml of methanol, and aqueous formaldehyde solution (4.21 g, 37% by mass) and aqueous sodium hydroxide solution (6.5 ml, 45.3% by mass) were added dropwise. After the addition was complete, the temperature was raised to 60°C and stirred at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25°C and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry, filtered, and dried to obtain compound 2 (4.8 g, 85.9% yield) as a white solid. ESI-MS (m / z) = 260.2 [M+H] +.

[0250] (1.3.2) Synthesis of compound 3

[0251] Compound 2 (4.8 g), prepared in step (1.3.1), was dissolved in 25 ml of 1,4-dioxane. A 4 M solution of hydrochloric acid in 1,4-dioxane (25 ml) was added, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (3.6 g, 99.4% yield) as a white solid.

[0252] (1.3.3) Synthesis of Compound 11

[0253] Compound 3 (3.6 g) prepared in step (1.3.2) was dissolved in 36 ml of DMF, and TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g) were added, and the mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 200 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 100 ml of ethyl acetate (3 × 100 ml). The organic phases were combined, washed once with 50 ml of saturated aqueous sodium chloride solution (1 × 50 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to obtain compound 11 (2.3 g, yield 33.0%) as a white solid. ESIMS (m / z) = 379.5 [M+H] + .

[0254] (1.3.4) Synthesis of Compound 12

[0255] Compound 11 (2.3 g) prepared in step (1.3.3) was dissolved in 23 ml of methanol, and wet palladium on carbon (230 mg, 10% loading) was added. The atmosphere was replaced with hydrogen three times, and the reaction system was stirred at 25°C under a hydrogen atmosphere (15 psi) for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filtrate, which was evaporated to dryness under reduced pressure to obtain compound 12 (1.48 g, 99.8% yield) as a yellow oil.

[0256] (1.3.5) Synthesis of Compound 13

[0257] Compound 12 (1.48 g) prepared in step (1.3.4) was dissolved in 15 ml of DMF, and triethylamine (TEA, 1.22 g), compound 4 (1.35 g), and HBTU (3.45 g) were added. The mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 150 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 50 ml of ethyl acetate (3 × 50 ml). The organic phases were combined, washed once with 30 ml of saturated aqueous sodium chloride solution (1 × 30 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (C18 column, eluent: water / acetonitrile = 5 / 1, v / v) to obtain compound 13 (1.3 g, yield 31.8%) as a white solid. ESI-MS (m / z): 674.3 [M+H]+.

[0258] (1.3.6) Synthesis of Compound 14

[0259] Compound 13 (1.1 g) prepared in step (1.3.5) was dissolved in 11 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath, and DMTrCl (813 mg) was added portionwise at 0°C. The reaction system was stirred at 0°C for 1 hour. After the reaction, methanol was added to quench the reaction solution, the solvent was evaporated, and the product was purified by reverse phase column chromatography (eluent: water / acetonitrile = 1 / 4, v / v) to obtain compound 14 (800 mg, 50.3% yield) as a white solid. ESI-MS (m / z): 976.5 [M+H]+.

[0260] (1.3.7) Synthesis of compound CR01013

[0261] At 25°C, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM). 4,5-Dicyanoimidazole (DCl, 53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 255.4 mg) were added. The atmosphere was purged with nitrogen three times, and the reaction system was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed twice with 5 ml of saturated sodium bicarbonate solution (2 × 5 ml) and once with 30 ml of saturated sodium chloride solution (1 × 30 ml). The organic phase was separated and dried over anhydrous sodium sulfate. The organic phase solvent was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 20 / 1, v / v) to obtain compound CR01013 (532 mg, 80.4% yield) as a white solid. ESI-MS (m / z): 1176.7 [M+H]+.

[0262] 1 HNMR(400MHz, DMSO-d6)δ0.95–1.05(d,J=6.7Hz,5H),1.06–1.15(q,J=7.6Hz,8H),1.15–1.21(t,J=7.2Hz,14H),1.72– 1.80(s,3H),1.84–1.92(s,3H),1.94–2.07(d,J=16.0Hz,7H),2.07–2.14(s,3H),2.64–2.72(q,J=5.8Hz,2H),2.74–2.8 9(d,J=8.5Hz,2H),3.35–3.56(m,4H),3.57–3.70(m,4H),3.71–3.77(s,6H),3.81–3.93(m,1H),3.96–4.09(d,J=6.4Hz, 3H), 6.82–6.97 (d, J = 8.7Hz, 4H), 7.17–7.27 (t, J = 8.7Hz, 5H), 7.27–7.34 (t, J = 7.6Hz, 2H), 7.34–7.43 (d, J = 7.5Hz, 2H).

[0263] Preparation Example 4 Synthesis of Compound CR01013Z

[0264] The synthetic route of compound CR01013Z is as follows:

[0265] (1.4.1) Synthesis of Compound 15

[0266] Compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane at 25°C. Triethylamine (25.9 mg, 0.25 mmol), DMAP (1.25 mg, 0.01 mmol), and compound 8 (succinic anhydride, 15.4 mg, 0.15 mmol) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the solvent was evaporated and the product was purified by reverse phase column chromatography (C18 column, eluent: water / acetonitrile = 2 / 1, v / v) to obtain compound 15 (110 mg, 0.10 mmol, 100% yield) as a yellow oil. ESI-MS (m / z) = 1099.3 [M+Na]+.

[0267] (1.4.2) Synthesis of compound CR01013Z

[0268] Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 ml of acetonitrile, and HBTU (24.2 mg, 0.06 mmol), DIEA (11.0 mg, 0.08 mmol), and amino-CPG (1.06 g, loading 80 μmol / g) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filter cake, which was washed twice with 50 ml of dichloromethane (2 × 50 ml), three times with 50 ml of acetonitrile (3 × 50 ml), and once with 50 ml of ethyl acetate (1 × 50 ml), and then vacuum dried. Cap 1 (4.8 ml), Cap 2 (0.54 ml), and DMAP (2.59 mg) were added to the dried filter cake, and the reaction system was stirred at 25°C for 5 hours. After the reaction was completed, the reaction solution was filtered to obtain a filter cake, which was washed three times with 50 ml of acetonitrile (3×50 ml) and dried in vacuo to obtain compound CR01013Z (900 mg, loading amount of 20-30 μmol / g).

[0269] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0270] Compound L96-PS

[0271] The structural formula of compound L96-PS is as follows:

[0272] Wherein, PS represents polystyrene resin solid phase carrier.

[0273] Preparation of double-stranded oligonucleotides (siRNA)

[0274] Preparation Example 5

[0275] (1.5.1) Synthesis of positive chain SS

[0276] Using phosphoramidite solid-phase nucleic acid synthesis, nucleoside monomers are linked one by one in a 3'-5' direction according to the nucleotide sequence. Each linking step involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are as follows:

[0277] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0278] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0279] The conditions for each coupling reaction were the same. The coupling reaction conditions were: temperature 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0280] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0281] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0282] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0283] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to a solid support was added to 0.5 ml / μmol of 25% ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone 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 trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0284] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the eluent ratio was eluent 1:eluent 2 (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0285] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC); molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured and theoretical values ​​were consistent, the compound was conjugated to the 3' end of the siRNA sense strand.

[0286] (1.5.2) Synthesis of antisense strand AS

[0287] The antisense strand was synthesized using a universal solid-phase support. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification, and desalting conditions in the solid-phase synthesis of the antisense strand were the same as those in step (1.5.1) for the synthesis of the sense strand.

[0288] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS, purchased from Waters, model: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured and theoretical values ​​were consistent, the siRNA antisense strand was obtained.

[0289] (1.5.3) siRNA synthesis

[0290] The sense strand synthesized in step (1.5.1) and the antisense strand synthesized in step (1.5.2) are mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. The mixture is slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense strand and antisense strand to form a double-stranded structure through hydrogen bonds, thereby obtaining the target siRNA.

[0291] Detection: Each siRNA was diluted to a concentration of 0.2 mg / ml (based on siRNA) using ultrapure water (Milli-Q ultrapure water analyzer, resistivity 18.2 MΩ*cm (25°C)). Molecular weights were then determined using liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The measured values ​​were consistent with the theoretical values, demonstrating that the synthesized siRNAs were the intended double-stranded nucleic acid sequences.

[0292] Preparation of double-stranded oligonucleotide (siRNA) conjugates

[0293] Preparation Example 6

[0294] (1.6.1) Synthesis of the positive chain

[0295] The method of phosphoramidite nucleic acid solid phase synthesis is used, starting with the above-mentioned compounds connected to the solid phase support (i.e., CR01008Z, CR01013Z, L96-PS), and the nuclei are connected one by one in a 3'-5' direction according to the nucleotide sequence (compounds CR01008 and CR01013 can be regarded as a nucleoside monomer). Each connection of a nucleoside monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the synthesis of the sense chain of this example are the same as those in the synthesis of the sense chain in step (1.5.1) of Preparation Example 5.

[0296] (1.6.2) Synthesis of antisense strand

[0297] The antisense strand was synthesized using a universal solid phase support. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis of the antisense strand were the same as those in step (1.5.2) of Preparation Example 5 for the synthesis of the sense strand.

[0298] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS, purchased from Waters, model: LCT Premier). The measured molecular weight was compared with the theoretical value. If the measured and theoretical values ​​were consistent, the siRNA antisense strand was obtained.

[0299] (1.6.3) siRNA synthesis

[0300] The sense chain synthesized in step (1.6.1) and the antisense chain synthesized in step (1.6.2) are 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 to allow the sense chain and antisense chain to form a double-stranded structure through hydrogen bonds, thereby obtaining the target siRNA conjugate.

[0301] Detection: Each siRNA was diluted to a concentration of 0.2 mg / ml (based on siRNA) using ultrapure water (Milli-Q ultrapure water analyzer, resistivity 18.2 MΩ*cm (25°C)). Molecular weights were then determined using liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The measured values ​​were consistent with the theoretical values, demonstrating that the synthesized siRNA conjugates contained the desired double-stranded nucleic acid sequence.

[0302] When the ligand is three clusters of CR01008, the structural formula of the siRNA conjugate is:

[0303] When the ligand is a triple cluster of CR01013, the structural formula of the siRNA conjugate is:

[0304] [Corrected 02.09.2024 according to Rule 26] When the ligand is L96, the structural formula of the siRNA conjugate is:

[0305] [Corrected 02.09.2024 in accordance with Rule 26] Among them, Indicates siRNA.

[0306] The sequences of the unmodified double-stranded oligonucleotides described in the present disclosure are shown in Table 1.

[0307] Table 1 Unmodified double-stranded oligonucleotide sequence list

[0308] The sequences of the modified double-stranded oligonucleotides disclosed in the present invention are shown in Table 2.

[0309] Table 2 Modified double-stranded oligonucleotide sequence list

[0310] In the present disclosure, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of a nucleotide; a lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; a lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; a lowercase letter d represents that the nucleotide adjacent to the left of the letter d is a deoxyribonucleotide; a lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0311] The information of the double-stranded oligonucleotide conjugate whose ligand is L96 described in the present disclosure is shown in Table 3.

[0312] Table 3 Information of double-stranded oligonucleotide conjugates with ligand L96

[0313] The information of the double-stranded oligonucleotide conjugates in which the ligands described in the present disclosure are three clusters of CR01008 is shown in Table 4.

[0314] Table 4 Information of double-stranded oligonucleotide conjugates with ligand (CR01008) × 3

[0315] Biological detection experiments

[0316] Unless otherwise stated, the reagents and consumables (Table 5) and instruments and equipment (Table 6) used in this application were commercially available products from the following manufacturers.

[0317] Table 5 Main reagents and consumables

[0318] Table 6 Main instruments and equipment

[0319] Unless otherwise specified, the human liver cancer cell line Huh7 used in the present disclosure was purchased from Wuhan Pronocell Life Science Co., Ltd.; the experimental animals C57BL / 6J mice used in the present disclosure were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; the experimental animals Balb / c mice used in the present disclosure were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.; the BKS-DB mice used in the present disclosure were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; the triglyceride and total cholesterol levels in the serum of the BKS-DB mice in the present disclosure were tested by Anling Biopharmaceutical (Suzhou) Co., Ltd.

[0320] In the context of this disclosure, unless otherwise stated, the real-time PCR detection data of the activity experiments involved in this disclosure are all calculated using the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0321] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0322] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0323] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0324] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0325] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0326] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0327] Test group target gene mRNA inhibition rate = 100% - test group target gene mRNA relative expression level

[0328] In the context of the present disclosure, unless otherwise stated, the in vivo activity experimental data are expressed as X±STDEV, and the experimental data are graphed and analyzed using GraphPad prism 8.0 software.

[0329] Example 1 Evaluation of siRNA activity in vitro

[0330] In this example, the target gene inhibitory activity evaluation method in the human liver cancer cell line Huh7 was used to evaluate the inhibitory activity of RX008001 to RX008154 on the target gene INHBE in cells at the same INHBE target site, with RX000001 as a negative control.

[0331] Test sample preparation:

[0332] After centrifugation of each siRNA test sample, add an appropriate amount of PBS according to the specifications of each tube to dissolve it and prepare a 20 μM stock solution. The stock solution is further diluted with PBS in a gradient manner to form 1 μM and 0.1 μM working solutions or 0.1 μM and 0.01 μM working solutions. Dosage tests are performed with final duplex concentrations of 10 nM and 1 nM or 1 nM and 0.1 nM.

[0333] 96-well transfection and detection:

[0334] Huh7 cells grown to near confluence were digested with trypsin, and the cells were washed to prepare a cell suspension. 100 μL of cell suspension was added to each well of a 96-well plate, with 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. After the cells adhered for 24 hours, the DMEM medium in the 96-well plate was aspirated and 80 μL of Opti-MEM was added to each well. TMThe 96-well plate was then placed in an incubator for further incubation. 1 μL of 1 μM, 0.1 μM, or 0.01 μM working solution was dispersed in 9 μL of Opti-MEM to form a siRNA mixture. 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and mixed with each siRNA mixture to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then the transfection complex was added to the 96-well plate at a rate of 20 μL / well. After 4 hours of incubation, 100 μL of DMEM medium supplemented with 20% FBS was added to each well, and the 96-well plate was placed in an incubator for an additional 24 hours.

[0335] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0336] Use reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 Reverse transcription primers, according to the method of reverse transcription kit specification sheet, configure 20 μ L reverse transcription system and complete reverse transcription reaction.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in HepG2 cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use primers for target gene and primers for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primers is shown in Table 7.

[0337] Table 7 Sequences of detection primers

[0338] In the real-time fluorescence quantitative PCR method, the ΔΔCt method is used according to the technical method described in the embodiment to perform relative quantitative calculation of the expression level of the target gene mRNA in each test group.

[0339] Table 8 Inhibitory activity of target genes in Huh7 cells at 10 nM and 1 nM doses after siRNA administration

[0340] Table 9 Inhibitory activity of target genes in Huh7 cells at 1 nM and 0.1 nM doses after siRNA administration

[0341] Example 2 Evaluation of the activity of L96-liganded siRNA conjugates in mice using the high-pressure hydrodynamic injection (HDI) model

[0342] In this example, the Balb / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of a conjugate containing the 3' end of the sense strand of the same INHBE target siRNA conjugated with the L96 ligand on the target gene INHBE.

[0343] Plasmid construction: pcDNA-CMV-RG008 plasmid (ID: NM_031479.5) was constructed by Sangon Biotech (Shanghai) Co., Ltd.

[0344] Mouse model construction:

[0345] The Balb / c mouse hydrodynamic injection model is established by rapidly injecting a pcDNA-CMV-RG008 plasmid solution through the tail vein at high pressure. On day 3 of the experiment, mice were injected with 10 μg of pcDNA-CMV-RG008 via the tail vein hydrodynamic injection over 5 seconds, at an injection volume of 8% of their body weight. The plasmid DNA for injection was diluted with normal saline, prepared immediately prior to injection, and stored at 4°C.

[0346] Animal grouping, drug administration and tissue sample collection:

[0347] 6-8 week old Balb / c mice (all female) were randomly divided into groups according to body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were dosed according to body weight, and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.1 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight. That is, the dosage of each drug conjugate was 1 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3). On the 4th day (recorded as D4), 5 mice of all groups were killed. The mice were subjected to gross dissection and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored with RNA later.

[0348] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0349] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo (dT) was selected. 18 Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription systems and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as an internal reference gene, and the target gene and the Nero internal reference gene were detected using primers for the target gene and primers for the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 10.

[0350] Table 10 Sequences of detection primers

[0351] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TMFast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer, 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level of the target gene mRNA in each test group was relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0352] Table 11 Inhibitory activity of target genes in Balb / c-HDI mice after administration of siRNA conjugates

[0353] As shown in Figure 1 and Table 11, at a dose of 1 mg / kg, RZ008003, RZ008019, RZ008020, RZ008023, RZ008026, and RZ008028 could significantly inhibit INHBE mRNA expression, with an inhibition rate of more than 70%.

[0354] Example 3 Activity Evaluation of siRNA Conjugates of L96 Ligands in C57BL / 6J Mice

[0355] This example evaluates the mRNA inhibitory activity of siRNA conjugates RZM08001-RZM08024, whose ligand is L96, in the liver tissue of C57BL / 6J mice, with RZ000001 as a negative control.

[0356] Animal grouping, drug administration and tissue sample collection:

[0357] 6-8 week old C57BL / 6J male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 5 mice per group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight, and the administration volume was 10 mL / kg of mouse body weight. Single administration was performed by subcutaneous injection in the abdomen, and each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution, i.e., the dosage of each drug conjugate was 3 mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate).

[0358] The day of administration was recorded as day 0 (recorded as D0), and all mice in the groups were killed on the 7th day after administration (recorded as D7). The mice were grossly dissected and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored in RNA later and INHBE mRNA expression levels were measured. In this real-time fluorescence quantitative PCR method, GAPDH gene was used as the internal reference gene, and primers for the target gene and the GAPDH internal reference gene were used for detection. The sequences of the detection primers are shown in Table 12.

[0359] Table 12 Sequences of detection primers

[0360] In the real-time fluorescence quantitative PCR method, the ΔΔCt method is used according to the technical method described in the embodiment to perform relative quantitative calculation of the expression level of the target gene mRNA in each test group.

[0361] Table 13 Inhibitory activity of target genes in liver tissue of C57BL / 6J mice after administration of siRNA conjugates

[0362] As shown in Figure 2 and Table 13, 7 days after a single subcutaneous administration of 3 mg / kg, RZM08015 and RZM08019 could significantly reduce the mRNA expression levels in the liver tissue of C57BL / 6J mice, with the mRNA inhibition effect of RZM08019 reaching 70%.

[0363] Example 4 Evaluation of the activity of siRNA conjugates with L96 ligand in C57BL / 6J mice

[0364] This example evaluates the mRNA inhibition activity of the L96 vector conjugate RZM08019 in the liver tissue of C57BL / 6J mice at different doses after single administration.

[0365] Animal grouping, drug administration and tissue sample collection:

[0366] 6-8 week old C57BL / 6J male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose was calculated for all mice according to body weight, and the administration volume was 10 mL / kg of mouse body weight. Single administration was performed by subcutaneous injection in the abdomen, and each drug conjugate was administered in the form of 0.9 mg / mL, 0.6 mg / mL, 0.3 mg / mL, 0.1 mg / mL and 0.03 mg / mL (calculated as siRNA) PBS solution, i.e., the dosage of each drug conjugate was 9 mg / kg, 6 mg / kg, 3 mg / kg, 1 mg / kg and 0.3 mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate).

[0367] The day of administration was recorded as day 0 (recorded as D0), and all mice in the groups were killed on the 7th day after administration (recorded as D7). The mice were grossly dissected and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored in RNA later and INHBE mRNA expression levels were measured. In this real-time fluorescence quantitative PCR method, GAPDH was used as an internal reference gene. Primers targeting the target gene and primers targeting the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively. The sequences of the detection primers are shown in Table 12.

[0368] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0369] Table 14 Inhibitory activity of target genes in C57BL / 6J mice after administration of different doses of RZM08019

[0370] The data results in Table 14 and Figure 3 show that RZM08019 can dose-dependently reduce the mRNA expression level in the liver tissue of C57BL / 6J mice.

[0371] Example 5 Evaluation of the efficacy of siRNA conjugate RZM08019 in BKS-DB mice

[0372] In this example, the target gene inhibitory activity evaluation method in mice was used to evaluate the inhibitory activity of RZM08019 on the target gene INHBE in mice; and the changes in triglycerides and total cholesterol in mouse serum were detected using an automatic biochemical analyzer to evaluate the lipid-regulating effect of RZM08019 in mice.

[0373] Animal grouping, drug administration and tissue sample collection:

[0374] 6-8 week old BKS-DB male mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were randomly divided into groups according to body weight, with 6 mice in the PBS group and 6 mice in the drug conjugate RZM08019 Q2W×2 group (administered once every two weeks, 2 times in total). The drug dose for all mice was calculated based on body weight, and the administration volume was 10 mL / kg mouse body weight. RZM08019 was repeatedly administered by subcutaneous injection in the abdomen, once every two weeks, for a total of 2 times. The drug conjugate was administered in the form of a PBS solution at 0.9 mg / mL (calculated as siRNA), that is, the dosage was 9 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as D0). The serum of mice in all groups was collected on the 7th day, 14th day (recorded as D14), 21st day (recorded as D21), 28th day (recorded as D28), and 35th day (recorded as D35) after administration and sent to Anling Biopharmaceutical (Suzhou) Co., Ltd. for detection of serum triglycerides and total cholesterol using a fully automatic biochemical detector.

[0375] All mice in the groups were killed on the 35th day after administration (denoted as D35). The killed mice were dissected and the liver tissues of each killed mouse were collected. The liver tissues were cut into approximately 2 mm 3 Small pieces were stored in RNA later and INHBE mRNA expression levels were measured. In this real-time fluorescence quantitative PCR method, GAPDH was used as an internal reference gene. Primers targeting the target gene and primers targeting the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively. The sequences of the detection primers are shown in Table 12.

[0376] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0377] Table 15 Triglyceride levels in serum of BKS-DB mice after repeated administration of RZM08019

[0378] Table 16 Total cholesterol levels in serum of BKS-DB mice after repeated administration of RZM08019

[0379] Table 17 INHBE mRNA expression levels in liver tissue of BKS-DB mice after repeated administration of RZM08019

[0380] The data results in Tables 15-17 and Figures 4-5 show that after subcutaneous injection of RZM08019 9 mg / kg Q2W×2 conjugate treatment in BKS-DB mice, RZM08019 can significantly reduce the CHO expression level in mouse serum; in experiment D35, it was observed that RZM08019 significantly reduced the mRNA expression level in mouse liver tissue, with an inhibitory effect of greater than 85%.

[0381] Example 5 In vitro activity evaluation of siRNA conjugates with CR01008×3 as ligand

[0382] This example uses the target gene inhibitory activity assessment method in the human liver cancer cell line Huh7 to evaluate the inhibitory activity of the CR01008-siRNA conjugate on the target gene INHBE in cells.

[0383] Test sample preparation:

[0384] After centrifugation of each siRNA test sample, add an appropriate amount of PBS according to the specifications of each tube to dissolve it and prepare a 20 μM stock solution. Then further dilute the stock solution with PBS in a gradient manner to a 0.1 μM working solution. The dosage test is performed with a final duplex concentration of 1 nM.

[0385] 96-well transfection and detection:

[0386] Huh7 cells grown to near confluence were digested with trypsin, and the cells were washed to prepare a cell suspension. 100 μL of cell suspension was added to each well of a 96-well plate, with 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. After the cells adhered for 24 hours, the DMEM medium in the 96-well plate was aspirated and 80 μL of Opti-MEM was added to each well. TM The 96-well plate was then placed in an incubator and incubated for further incubation. 1 μL of the 0.1 μM working solution was dispersed in 9 μL of Opti-MEM to form the siRNA mixture. 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and mixed with each siRNA mixture to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then 20 μL of the transfection complex was added to the 96-well plate at a rate of 20 μL per well. After 4 hours of incubation, 100 μL of DMEM medium supplemented with 20% FBS was added to each well, and the 96-well plate was placed in an incubator and incubated for an additional 24 hours.

[0387] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0388] Use reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 Reverse transcription primers, according to the method of reverse transcription kit specification sheet, configure 20 μ L reverse transcription system and complete reverse transcription reaction.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in HepG2 cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use primers for target gene and primers for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primers is shown in Table 7.

[0389] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0390] Table 18 Inhibitory activity of target genes in Huh7 cells at a dose of 1 nM after administration of siRNA conjugates

[0391] The data results in Table 18 and FIG6 show that the siRNA conjugate provided in this example can significantly inhibit the expression level of INHBE mRNA in Huh7 cells.

[0392] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. A double-stranded oligonucleotide targeting the INHBE gene, characterized in that: The double-stranded oligonucleotide includes a sense strand and an antisense strand, wherein the antisense strand is complementary or substantially complementary to the sense strand; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides; wherein the sense strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence, and the substantially identical means that there is no more than 3 nucleotide difference between the sense strand and at least 15 consecutive nucleotides in the SEQ ID NO:309 sequence.

2. The double-stranded oligonucleotide according to claim 1, characterized in that The positive strand comprises a nucleotide sequence that differs from at least 15 consecutive nucleotides in the sequence of SEQ ID NO: 309 by no more than 2 nucleotides, preferably by no more than 1 nucleotide.

3. The double-stranded oligonucleotide according to claim 1, characterized in that The antisense strand is complementary or substantially complementary to at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 309, wherein the substantially complementary strand has no more than 3 nucleotide mismatches in the complementary region.

4. The double-stranded oligonucleotide according to claim 1, characterized in that The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 consecutive nucleotides of any one of the sequences shown in SEQ ID NO:155 to SEQ ID NO:308 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; The sense strand comprises a nucleotide sequence that is at least partially reverse complementary or substantially complementary to the antisense strand to form a double-stranded region; the substantially complementary sequence means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides.

5. The double-stranded oligonucleotide according to claim 4, characterized in that The antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 3 nucleotides; Optionally, the antisense strand comprises at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 2 nucleotides; Optionally, the antisense strand comprises at least 19, at least 20, or at least 21 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NO: 155 to SEQ ID NO: 308 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 1 nucleotide; Optionally, the antisense strand is selected from or comprises any one of the nucleotide sequences shown in SEQ ID NO.155 to SEQ ID NO.308 in Table 1; Optionally, based on the 5'-3' direction, positions 2-19 of the antisense strand comprise at least 15 nucleotides of the nucleotides 2-19 of any one of the nucleotide sequences shown in SEQ ID NO.155 to SEQ ID NO.308 in Table 1, or a nucleotide sequence that differs from the at least 15 nucleotides by less than 3 nucleotides.

6. The double-stranded oligonucleotide according to claim 4, characterized in that The sense strand comprises at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 3 nucleotides; Optionally, the sense strand comprises at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1; Optionally, the sense strand is selected from or comprises any one of the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.154 in Table 1.

7. The double-stranded oligonucleotide according to claim 4, characterized in that Each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides; Optionally, substantially all of the nucleotides of the sense strand or the antisense strand are selected from modified nucleotides; Optionally, all nucleotides of the sense strand and the antisense strand are selected from modified nucleotides.

8. The double-stranded oligonucleotide according to claim 7, characterized in that The modified nucleotides are each independently selected from 2'-halo, 2'-deoxy, 2'-O-(CH2) n -R1 modified nucleotide, or quasi-nucleotide; the quasi-nucleotide is selected from one or more of PNA, MNA, BNA, LNA, GNA, TNA and UNA; n is selected from 0, 1 or 2; R1 is selected from optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkoxy or -Si(R 1a )3; Each R 1a are independently selected from optionally substituted C 1-6 Alkyl or optionally substituted C 1-6 Alkoxy; Optionally, 2'-O-(CH2) n -R1 is selected from 2'-O-CH3, 2'-O-CH2-O-CH3, 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3 or 2'-O-CH2-CH2-O-CH3.

9. The double-stranded oligonucleotide according to claim 4, characterized in that The sense strand or the antisense strand comprises a 3' overhang having at least 1 nucleotide; Optionally, the antisense strand comprises a 3' overhang of at least 1 nucleotide; Optionally, the sense strand or the antisense strand comprises a 3' overhang of at least 2 nucleotides; Optionally, the antisense strand comprises a 3' overhang of at least 2 nucleotides; Optionally, the antisense strand comprises a 3' overhang of 2 nucleotides; Optionally, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds; Optionally, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end; Optionally, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

10. The double-stranded oligonucleotide according to any one of claims 1 to 9, characterized in that All nucleotides of the sense strand and all nucleotides of the antisense strand are selected from modified nucleotides; wherein the double-stranded region formed by the sense strand and the antisense strand is as shown in the following formula (I): SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5'(I), Among them, SS represents the sense strand and AS represents the antisense strand; Each N is independently selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-deoxy modified nucleotide; Each X is independently selected from 2'-O-TBDMS, 2'-O-TIPS, 2'-O-TOM, 2'-O-CH2-O-CH2-CH3, 2'-O-CH2-O-CH2-CF3, 2'-O-CH2-CH2-O-CH3; Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10; and p', q', r', p, q are not 0 at the same time, and 0≤q'+r'≤4; Optionally, a' is selected from an integer of 3-8, p' is selected from 0 or 1, b' is selected from an integer of 4-13, q' is selected from 0 or 1, c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, p is selected from 1, b is selected from an integer of 4-8, q is selected from 0 or 1, and c is selected from an integer of 6-10; Optionally, each N is independently selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides; Optionally, each X is independently selected from 2'-O-methyl modified nucleotides, 2'-O-MOE (methoxyethyl) modified nucleotides, 2'-O-TBDMS modified nucleotides, 2'-O-TIPS modified nucleotides, 2'-O-TOM modified nucleotides, 2'-O-CH2-O-CH2-CH3 modified nucleotides, 2'-O-CH2-O-CH2-CF3 modified nucleotides; Preferably, each X is independently selected from 2'-O-methyl modified nucleotides, 2'-O-MOE modified nucleotides, 2'-O-CH2-O-CH2-CH3 modified nucleotides, 2'-O-CH2-O-CH2-CF3 modified nucleotides; Optionally, the double-stranded region comprises at least one 2'-O-methoxyethyl modified nucleotide or 2'-O-ethoxymethyl modified nucleotide.

11. The double-stranded oligonucleotide according to claim 10, characterized in that In the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide; Optionally, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl modified nucleotide, at least four of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl modified nucleotide, at least five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl modified nucleotide, any five of the nucleotides at positions 2, 6, 9, 12, 14, and 16 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from a 2'-O-methoxyethyl-modified nucleotide, the nucleotides at positions 2, 6, 9, 14, and 16 are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotide at position 15 of the antisense strand is selected from 2'-O-methoxyethyl modified nucleotides, the nucleotides at positions 2, 6, 12, 14, and 16 are selected from 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides.

12. The double-stranded oligonucleotide according to claim 10, characterized in that In the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7 to 10 of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

13. The double-stranded oligonucleotide according to claim 10, characterized in that The antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of any modified antisense strand nucleotide sequence shown in Table 2, or a nucleotide sequence that differs from the consecutive nucleotides by no more than 3 nucleotides; Optionally, the antisense strand comprises at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of any modified antisense strand nucleotide sequence shown in Table 2; Optionally, the antisense strand is selected from or comprises any one of the modified antisense strand nucleotide sequences shown in Table 2.

14. The double-stranded oligonucleotide according to claim 10, characterized in that The sense strand comprises at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of any modified sense strand nucleotide sequence shown in Table 2, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides; Optionally, the sense strand of the double-stranded oligonucleotide is selected from or comprises any modified sense strand nucleotide sequence shown in Table 2.

15. The double-stranded oligonucleotide according to claim 10, characterized in that The sense strand of the double-stranded oligonucleotide is selected from or comprises any modified sense strand nucleotide sequence shown in Table 2; the antisense strand is selected from or comprises any modified antisense strand nucleotide sequence shown in Table 2.

16. The double-stranded oligonucleotide according to any one of claims 1 to 15, characterized in that The double-stranded oligonucleotide is selected from siRNA.

17. A double-stranded oligonucleotide conjugate, characterized in that The conjugate comprises the double-stranded oligonucleotide of any one of claims 1 to 15 and one or more ligands capable of binding to a cell receptor; Optionally, the ligand is conjugated to the sense strand and / or the antisense strand; Optionally, the ligand is conjugated to the 3' end and / or the 5' end of the sense strand; Optionally, the ligand is conjugated to the 3' end of the sense strand; Optionally, the cell receptor is selected from asialoglycoprotein receptors; Optionally, the ligand comprises galactose or a cluster of galactose.

18. The conjugate according to claim 17, characterized in that The ligand is selected from the structure shown in formula (101) or its isomers or pharmaceutically acceptable salts thereof: Wherein, * represents the conjugation site of the ligand with the sense strand or the antisense strand; m is selected from 1, 2, 3 or 4; Each Z is independently selected from hydroxyl or thiol; Each p is independently selected from 1, 2 or 3; Each q is independently selected from 1, 2 or 3; Each R is independently selected from H, optionally substituted C1-C6 alkyl, or optionally substituted C1-C6 alkoxy; Each L is independently selected from an optionally substituted C2-C20 alkylene or R La and R La independently selected from optionally substituted C1-C10 alkylene, k is selected from 1, 2, 3, 4 or 5; Each Y is independently selected from O, S or NH; Optionally, m is selected from 1, 2 or 3; Optionally, Z is selected from hydroxyl; Optionally, p is selected from 1; Optionally, q is selected from 1; Optionally, R is selected from H; Optionally, each L is independently selected from C1-C10 alkylene or Among them, R La and R La Independently selected from C1-C5 alkylene, k is 1, 2 or 3; Optionally, k is selected from 1; Optionally, each L is independently selected from Optionally, Y is selected from O.

19. The conjugate according to claim 17, characterized in that The ligand is selected from the following structures or isomers thereof or pharmaceutically acceptable salts thereof:

20. The conjugate according to claim 17, characterized in that The ligand is selected from the following structures or isomers thereof or pharmaceutically acceptable salts thereof: Wherein, * represents the conjugation site of the ligand with the sense strand or the antisense strand.

21. A composition comprising any of the following: (I) the double-stranded oligonucleotide according to any one of claims 1 to 16; and / or (II) The conjugate according to any one of claims 17 to 20.

22. Use of any of the following in the preparation of a medicament for preventing and / or treating a disease or condition mediated by the INHBE gene: (I) the double-stranded oligonucleotide according to any one of claims 1 to 16; and / or (II) the conjugate according to any one of claims 17 to 20; and / or (III) the composition of claim 21; Optionally, the disease or condition includes, but is not limited to, having or being at risk for developing a metabolic disorder, type 2 diabetes, obesity, elevated triglyceride levels, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes, non-alcoholic steatohepatitis (NASH), cardiovascular disease, cardiomyopathy, hypertension, and / or heart failure.

23. A pharmaceutical composition comprising any of the following and a pharmaceutically acceptable excipient or adjuvant: (I) the double-stranded oligonucleotide according to any one of claims 1 to 16; and / or (II) the conjugate according to any one of claims 17 to 20; and / or (III) The composition according to claim 21.

24. A method for reducing the expression or activity of the INHBE gene, characterized in that: It comprises contacting a cell with any of the following: (I) the double-stranded oligonucleotide according to any one of claims 1 to 16; and / or (II) the conjugate according to any one of claims 17 to 20; and / or (III) the composition of claim 21; and / or (IV) The pharmaceutical composition of claim 23.

25. A method for preventing and / or treating a disease or condition mediated by the INHBE gene, characterized in that: It comprises administering to a subject a pharmaceutically acceptable amount of any of the following: (I) the double-stranded oligonucleotide according to any one of claims 1 to 16; and / or (II) the conjugate according to any one of claims 17 to 20; and / or (III) the composition of claim 21; and / or (IV) The pharmaceutical composition of claim 23.

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