Inhbe gene-targeting oligonucleotide and use thereof

Abstract

Provided are an INHBE gene-targeting oligonucleotide and a use thereof. The provided oligonucleotide can reduce the level of INHBE in vivo, and acts as an inhibitor of INHBE.

Description

Oligonucleotides targeting the INHBE gene and uses thereof TECHNICAL FIELD

[0001] The present disclosure relates to an oligonucleotide, in particular to the inhibition of the expression of the inhibin subunit beta E (INHBE) gene and the treatment or prevention of INHBE-related disorders, conditions and / or pathologies by inhibition of INHBE. BACKGROUND

[0002] Nowadays, obesity has become one of the killers endangering human health. According to the data of the World Health Organization, overweight and obesity are the sixth leading cause of death globally, with at least 3.4 million people dying each year as a result of overweight or obesity. The distribution of adipose tissue plays an important role in metabolic health and cardiovascular disease risk: obesity can easily lead to a series of cardiovascular system diseases such as fatty liver, hypertension, coronary heart disease, and affect the metabolism of sugar in the body. Excessive obesity can easily lead to abnormal glucose tolerance or the occurrence of diabetes to trigger metabolic syndrome.

[0003] Among all obese people, those with a characteristic of more abdominal fat, such as "beer belly" and "swimming ring", face higher health risks. Both epidemiological and genetic studies have shown that the waist-to-hip ratio (WHR) adjusted for body mass index (BMI) has an important impact on cardiometabolic diseases and mortality. A Mendelian randomization study has determined that an increase in waist-to-hip ratio is causally associated with type II diabetes, coronary heart disease risk, and blood glucose traits, circulating lipids, and blood pressure.

[0004] While waist circumference can be adjusted through diet and exercise, we find that some people seem to be born with lower waist-to-hip ratios and "beer bellies" are hard to come by. Deaton et al. used whole-exome sequencing data from the UK Biobank to perform genetic analyses on 360,000 people. They found that INHBE contains a pLOF variant (Predicted loss of function, pLOF) associated with lower waist-to-hip ratios, with an average of one in 587 carriers. This mutation results in a 90% reduction in the levels of activin E, a cytokine secreted by the liver. Heterozygous carriers of the INHBE pLOF variant have good metabolic profiles, including lower triglyceride levels, higher "good cholesterol" - high-density lipoprotein cholesterol levels, and low levels of alanine aminotransferase and fasting glucose. The researchers further analyzed the survival of INHBE pLOF carriers using Cox proportional hazards regression and age data from a subset of exome-sequenced participants, and found no evidence of higher mortality rates among INHBE pLOF mutation carriers or adverse effects associated with INHBE pLOF. Drugs targeting INHBE could have a significant physiological effect by reducing abdominal fat, with a different biological mechanism than existing drugs for treating coronary heart disease and type II diabetes, and thus would be a powerful complement to existing drugs.

[0005] Inhibin subunit beta E (INHBE) is a member of the TGF-beta (transforming growth factor beta) superfamily of proteins. Inhibins are involved in the regulation of a variety of cellular processes, including cell proliferation, apoptosis, immune response, and hormone secretion. Inhibins and activins inhibit and stimulate, respectively, the secretion of follicle-stimulating hormone from the pituitary. Inhibin / activin, depending on its subunit composition, is involved in the regulation of a variety of different functions, such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythroid differentiation, insulin secretion, neural cell survival, embryonic axial development, or bone growth. Inhibins appear to function in opposition to activins. In addition, INHBE can be upregulated under endoplasmic reticulum stress conditions, and the protein can inhibit cell proliferation and growth in the pancreas and liver.

[0006] Body fat distribution is an important risk factor for cardiovascular and metabolic disease, independent of overall obesity. Body fat distribution characterized by more fat accumulation around the waist (such as more abdominal fat or a larger waist circumference) and / or less fat accumulation around the hips (such as less gluteofemoral fat or a smaller hip circumference), resulting in a larger waist-to-hip ratio (WHR), is associated with higher cardiovascular metabolic risk, independent of body mass index (BMI). Metabolic conditions associated with body fat distribution include, but are not limited to, type II diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid fractions), obesity (particularly abdominal obesity), lipodystrophy (such as an inability to deposit fat locally in fat depots (partial lipodystrophy) or an inability to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenge, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis, or liver inflammation), nonalcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, other markers of inflammation or fat deposition in the liver higher or elevated or altered, higher blood pressure and / or hypertension, higher blood glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the above conditions. Identifying genetic variants associated with a more favorable fat distribution (such as a lower WHR, particularly when adjusted for BMI) can be a pathway to identifying mechanisms that can be therapeutically used to benefit these cardiovascular metabolic diseases (Deaton, A. M., et al., Nature Communications, 2022. 13(1). SUMMARY

[0007] An object of the present disclosure is to provide an inhibitor of INHBE expression which is effective, safe, and has a long-lasting effect.

[0008] The present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof and a method of using the oligonucleotide or the pharmaceutically acceptable salt thereof to inhibit expression of an INHBE gene in a cell or a mammal, wherein the oligonucleotide targets the INHBE gene. Also provided herein are compositions and methods for treating pathological conditions and diseases in a mammal caused by expression of an INHBE gene. The oligonucleotide is a double-stranded RNA (dsRNA) that directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

[0009] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting INHBE expression, the oligonucleotide comprising a sense strand having a sequence with at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NOs. 3-167, or a fragment thereof, or a modified sequence thereof, preferably having 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity; and an antisense strand having a sequence with at least 60% or more sequence identity to a sequence set forth in any one of SEQ ID NOs. 170-334, or a fragment thereof, or a modified sequence thereof, preferably having 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

[0010] In another aspect, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting INHBE expression, comprising: (i) the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

[0011] In another aspect, the present disclosure provides a composition comprising the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or the conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0012] In another aspect, the present disclosure provides use of the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or the conjugate or a pharmaceutically acceptable salt thereof, or the composition in the manufacture of a medicament for treating or / and preventing an INHBE-related disorder, disease and / or condition.

[0013] In another aspect, the present disclosure provides a method for treating and / or preventing an INHBE-related disorder, disease and / or condition in a subject by administering a therapeutic agent (e.g., an oligonucleotide or a vector or transgene encoding the same) to the subject.

[0014] In another aspect, the present disclosure provides a method of using the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or the conjugate or the composition in combination with other drugs and / or other therapeutic methods for treating and / or preventing an INHBE-related disorder, disease and / or condition in a subject.

[0015] Metabolic conditions associated with body fat distribution that can be treated or prevented by inhibiting expression of INHBE include, but are not limited to, obesity, type II diabetes, hyperlipidemia or dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid moieties), obesity (particularly abdominal obesity), lipodystrophy (such as an inability to deposit fat locally in fat depots (partial lipodystrophy) or an inability to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenge, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis, or liver inflammation), nonalcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, higher or elevated or altered other markers of inflammation or fat deposition in the liver, higher blood pressure and / or hypertension, higher blood glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the above conditions.

[0016] Experiments demonstrate that the oligonucleotides of the present disclosure can effectively reduce INHBE content in vivo, and are effective inhibitors of INHBE. BRIEF DESCRIPTION OF DRAWINGS

[0017] FIG. 1 shows a diagram of siRNA solid phase synthesis.

[0018] FIG. 2 shows the results of in vivo testing of INHBE RNAi agents in transgenic mice. DETAILED DESCRIPTION

[0019] In the present disclosure, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be generally understood by one of ordinary skill in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and immunology, and laboratory operations procedures used herein are terms and procedures that are generally used in the respective fields. Also, for better understanding of the present disclosure, the definitions and explanations of the relevant terms are provided below.

[0020] As used herein, the term “about” or “approximately,” as applied to one or more target values, refers to a value that is similar to a reference value. In some embodiments, unless otherwise stated or otherwise evident from the context, the term “approximately” or “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the stated reference value in either direction (greater than or less than) unless such number would exceed 100% of a possible value.

[0021] As used herein, the term "complementary" refers to the structural relationship between nucleotides (e.g., on opposite nucleic acids or on opposite regions of a single nucleic acid strand) that allows the nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposite nucleic acid can base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows for the formation of a stable duplex. In some embodiments, two nucleic acids can have nucleotide sequences that are complementary to one another so as to form a region of complementarity, as described herein.

[0022] As used herein, the term "strand" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, a strand has two free ends, e.g., a 5'-end and a 3'-end.

[0023] As used herein, the term "deoxyribonucleotide" refers to a nucleotide having a hydrogen at the 2' position of its pentose sugar as compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions (including modifications or substitutions in or of the sugar, phosphate group, or base or modifications or substitutions of the sugar, phosphate group, or base) other than at the 2' position.

[0024] As used herein, the term "oligonucleotide" refers to a short nucleic acid, e.g., a short nucleic acid less than 100 nucleotides in length. An oligonucleotide can comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, e.g., modified ribonucleotides. An oligonucleotide can be single-stranded or double-stranded. An oligonucleotide can or can not have a duplex region. As a non-limiting example, an oligonucleotide can be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

[0025] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in duplex form. In some embodiments, one or more duplex regions of a double-stranded oligonucleotide are formed by complementary base pairing between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, one or more duplex regions of a double-stranded oligonucleotide are formed by complementary base pairing between antiparallel sequences of nucleotides of covalently linked nucleic acid strands. In some embodiments, one or more duplex regions of a double-stranded oligonucleotide are formed by complementary base pairing from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with each other. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed, e.g., with overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and thus, can have one or more mismatches, which can include internal mismatches or terminal mismatches.

[0026] As used herein, the terms "iRNA," "RNAi agent," "iRNA agent," "RNA interference agent" are used interchangeably herein to refer to an agent comprising the RNA as defined herein and that mediates the targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the sequence-specific degradation of mRNA. RNAi modulation, e.g., inhibition of INHBE expression in a cell, e.g., a cell in a subject, e.g., a mammalian subject.

[0027] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientation with respect to a target RNA (i.e., an INHBE gene). In some embodiments of the disclosure, double-stranded RNA (dsRNA) triggers the degradation of a target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally, most of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, either or both strands can also comprise one or more non-ribonucleotides, e.g., deoxyribonucleotides or modified nucleotides. In addition, as used herein, an "iRNA" can comprise ribonucleotides having chemical modifications; an iRNA can comprise substantial modifications at multiple nucleotides.

[0028] As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleotide" encompasses substitutions, additions, or removals, e.g., functional groups or atoms, to the internucleosidic linkage, sugar moiety, or nucleobase. Modifications suitable for agents of the present disclosure include all types of modifications disclosed herein or known in the art.

[0029] As used herein, "conjugate" refers to the linkage of two or more chemical moieties each having a specific function to one another in a covalent linkage; correspondingly, "conjugate" refers to a compound formed by the covalent linkage between the respective chemical moieties. Further, "siRNA conjugate" denotes a compound formed by the covalent linkage of one or more chemical moieties having a specific function to an siRNA. Hereinafter, the siRNA conjugate of the present disclosure is sometimes also referred to simply as "conjugate". The siRNA conjugate should be understood in context as a general term for siRNA conjugate, a first siRNA conjugate or a second siRNA conjugate, or an siRNA sense strand conjugate or an siRNA antisense strand conjugate.

[0030] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that overhangs from the duplex structure of a double-stranded iRNA. For example, a nucleotide overhang is present when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang can include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0031] As used herein, the term "naked sequence" refers to an unmodified nucleotide sequence.

[0032] As used herein, the term "inhibit" is used interchangeably with "knock down," "reduce," "silence," "down-regulate," "suppress," and other similar terms, and includes any degree of inhibitory effect.

[0033] The phrase "inhibiting expression of INHBE" is intended to refer to inhibiting expression of any INHBE gene, such as, for example, a mouse INHBE gene, a rat INHBE gene, a monkey INHBE gene, or a human INHBE gene, as well as variants or mutants of the INHBE gene. Thus, in the context of genetically manipulating a cell, population of cells, or organism, the INHBE gene can be a wild-type INHBE gene, a mutant INHBE gene, or a transgenic INHBE gene.

[0034] "Inhibiting expression of an INHBE gene" includes inhibition of an INHBE gene at any level, for example, at least partial inhibition of expression of an INHBE gene. Expression of an INHBE gene can be assessed based on the level or change in level of any variable associated with expression of an INHBE gene, for example, mRNA level or INHBE protein level, or indirectly by inhibiting mRNA level of a Gluc and INHBE fusion protein gene, which in turn reflects inhibition of INHBE protein level by inhibiting level of Gluc protein.

[0035] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids.

[0036] The term "pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable. These salts are prepared from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, particularly hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein. In addition, these salts can be prepared as base salts with inorganic bases or organic bases. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (e.g. sodium salt, potassium salt, and lithium salt), ammonium salt, alkaline earth metal salts (e.g. calcium salt and magnesium salt). Salts derived from organic bases include, but are not limited to, salts of a primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion-exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. The oligonucleotides of the present disclosure can also exist in a zwitterionic form. Particularly preferred pharmaceutically acceptable salts of the present disclosure are sodium, lithium, potassium and trialkylammonium salts.

[0037] As used herein, the term "subject" is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey and a chimpanzee), a non-primate (such as a cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or a bird, that endogenously or heterologously expresses a target gene. In one embodiment, the subject is a human.

[0038] As used herein, the term "treating" or "treatment" refers to an advantageous or desirable result, such as a reduction in at least one sign or symptom of an INHBE-associated disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesirable INHBE expression; lessening the extent of undesirable INHBE activation or stabilization; ameliorating or palliating undesirable INHBE activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesirable INHBE expression. "Treatment" can also mean prolonging survival as compared to the expected survival without treatment.

[0039] As used herein, the term "prevention" or "preventing" when used in reference to a disease or disorder, will benefit from a reduction in INHBE gene expression or INHBE protein production.

[0040] As used herein, the term "therapeutically effective amount" is intended to encompass an amount of an RNAi agent that is sufficient, when administered to a subject having an INHBE-associated disorder, to affect treatment of the disease (e.g., by reducing, ameliorating, or maintaining existing disease or one or more symptoms of the disease). The "therapeutically effective amount" can vary depending on the RNAi agent, how the agent is administered, the disease and its severity and history, the age, weight, family history, genetic makeup, type of prior or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0041] As used herein, the term "prophylactically effective amount" is intended to encompass an amount of an RNAi agent that is sufficient, when administered to a subject having an INHBE-associated disorder, to prevent or ameliorate the disorder or one or more symptoms of the disorder. Ameliorating a disease includes slowing the progression of the disease or reducing the severity of the disease that develops later. The "prophylactically effective amount" can vary depending on the RNAi agent, how the agent is administered, the extent of risk of the disease, and the history, age, weight, family history, genetic makeup, type of prior or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0042] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involving the delivery of the subject compound from one organ, or portion of the body, to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Such carriers are well known in the art. Pharmaceutically acceptable carriers include those used for injection.

[0043] This level can be assessed in an individual cell or cell population, including, for example, a sample from a subject. It can be appreciated that INHBE is primarily expressed in the liver, but also in the intestine, kidney, and brain, and is present in circulation.

[0044] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with INHBE expression compared to a control level. The control level can be any type of control level used in the art, for example, a pre-dose baseline level, or a level determined from a similar subject that has not been treated or treated with a control, such as, for example, a buffer control or a non-active agent control only.

[0045] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting INHBE expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having a sequence with at least 60% or more sequence identity to the sequence set forth in any one of SEQ ID NO. 3-167 or a fragment thereof or a modified sequence thereof, preferably having a sequence with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity; the antisense strand having a sequence with at least 60% or more sequence identity to the sequence set forth in any one of SEQ ID NO. 170-334 or a fragment thereof or a modified sequence thereof, preferably having a sequence with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

[0046] In some embodiments, the sense strand and / or the antisense strand is 15-30, 17-25, or 19 to 25 nucleotides in length.

[0047] In some embodiments, wherein each strand is independently 15-30, 17-25, or 19 to 25 nucleotides in length.

[0048] In some embodiments, the antisense strand is 19 to 23 nucleotides in length.

[0049] In some embodiments, the sense strand is 19 to 23 nucleotides in length.

[0050] In some embodiments, the oligonucleotide comprises a 5' and / or 3'- overhang sequence of one or more nucleotides in length, wherein the 5' and / or 3'- overhang sequence is present on the antisense strand and / or the sense strand. In one embodiment, the antisense strand of the oligonucleotide has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' end or the 5' end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' end or the 5' end. In another embodiment, one or more of the nucleotides in the overhang are replaced by a nucleoside thiophosphate.

[0051] In some embodiments, the antisense strand bears 1 or 2 overhangs.

[0052] In some embodiments, the sense strand bears 1 or 2 overhangs.

[0053] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of 1 or 2 nucleotides in length.

[0054] In some embodiments, the oligonucleotide comprises a 5'-overhang sequence of 1 or 2 nucleotides in length.

[0055] In some embodiments, the 3'-overhang sequence is present on the sense strand; preferably, the overhang sequence is selected from the group consisting of: GG, GA, GC, UC, UG, UU, UA, CA, CC, CG, CU, AA, AG, AU, AC.

[0056] In some embodiments, the 3'-overhang sequence is present on the antisense strand; preferably, the overhang sequence is selected from the group consisting of: UU, UC, UA, UG, GA, GG, GU, GC, TT, AG, AU, AA, AC, CA, CC, U; more preferably, the overhang sequence is UU.

[0057] In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand each ranging from 19 to 23 nucleotides in length.

[0058] In some embodiments, the sense strand forms a duplex region with the antisense strand.

[0059] In some embodiments, the sense strand and the antisense strand are 19 / 21-paired, 21 / 21-paired, 21 / 23-paired, or 23 / 23-paired duplex structures, respectively.

[0060] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0061] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0062] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0063] In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0064] In some embodiments, the pharmaceutically acceptable salt of the oligonucleotide can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium salt, potassium salt, and lithium salt), ammonium salt, alkaline earth metal salts (such as calcium salt and magnesium salt). Salts derived from organic bases (e.g., organic amines) include, but are not limited to, salts formed with primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.

[0065] In some embodiments, examples of the pharmaceutically acceptable salt of the oligonucleotide include, but are not limited to, ammonium salt, such as a salt of a tertiary alkylamine compound (e.g., a triethylamine salt), metal salt, such as a sodium salt, a potassium salt, and a magnesium salt, and the like.

[0066] In some embodiments, the oligonucleotide or salt thereof can be in the form of a hydrate or solvate.

[0067] In some embodiments, the oligonucleotide comprises at least one modified nucleotide.

[0068] In some embodiments, at least one of the modified nucleotides in the modified nucleotide is selected from the group consisting of: a deoxy nucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a 2'-5'-linked ribonucleotide (3'-RNA), an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-0-allyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyrane modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate analog, a vinyl-phosphonate nucleotide, a thermally unstable nucleotide, a glycol modified nucleotide, a nucleotide comprising a 2' phosphate, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

[0069] In some embodiments, at least one of the modified nucleotides in the modified nucleotide is selected from the group consisting of: a LNA modified nucleotide, a HNA modified nucleotide, a CeNA modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a 2'-0-allyl modified nucleotide, a 2'-C-allyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a 2'-hydroxyl modified nucleotide, and a glycol modified nucleotide; and combinations thereof.

[0070] In some embodiments, at least one of the modified nucleotides in the modified nucleotide is selected from the group consisting of: a deoxy nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a glycol modified nucleotide (GNA), a nucleotide comprising a 2' phosphate, and a nucleotide comprising a phosphorothioate group; and combinations thereof.

[0071] In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleotide.

[0072] In some embodiments, the 2'-modified nucleotides are selected from the group consisting of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-acylamido modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-allyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxynucleotides, nucleotides including 2' phosphate, and 2'-O-(N-methylacetamide) modified nucleotides; and combinations thereof. For example, C1-C3 alkoxy (e.g., methoxy); substituted alkoxy (e.g., C1-C3 alkoxy substituted C1-C3 alkoxy, e.g., methoxyethoxy); alkyl (e.g., C1-C3 alkyl, e.g., methyl), substituted alkyl (e.g., C1-C3 alkoxy substituted C1-C3 alkyl, e.g., methoxymethyl, methoxyethyl); amino (-NH2), substituted amino (e.g., C1-C3 alkyl mono or di-substituted amino, e.g., methylamino, ethylamino), but are not limited thereto.

[0073] In some embodiments, the 2'-modified nucleotides are selected from the group consisting of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxynucleotides; and combinations thereof.

[0074] In some embodiments, the 2'-modification is selected from the group consisting of 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl modifications; and combinations thereof.

[0075] In some embodiments, the 2'-modification is a 2'-methoxy modification.

[0076] In some embodiments, the 2'-modification is a 2'-acetamido modification.

[0077] In some embodiments, all of the nucleotides of the oligonucleotide are modified.

[0078] In some embodiments, the oligonucleotide comprises a modification at the 5' terminus comprising a 5'-phosphate analog or a 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine).

[0079] In some embodiments, the oligonucleotide has a 5'-phosphate analog modified nucleotide at the 5' terminal nucleotide.

[0080] In some embodiments, the 5'-phosphate analog modification is a 5'-(E)- vinylphosphonate (5'-VP).

[0081] In some embodiments, the modification is a modification selected from the group consisting of: 5'-phosphate analog modification, 2'-methoxy (CH3O-), 2'-fluoro (f), 2'-acetamido (CH3CO-NH-), and phosphorothioate (s).

[0082] In some embodiments, the oligonucleotide comprises a 6-(3-(2- carboxyethyl)phenyl) purine modified nucleotide at the 5' terminus. In some embodiments, the oligonucleotide comprises Formula M, which is a 2'-O-methyl-6-(3-(2- carboxyethyl)phenyl)-purine nucleotide as shown in Formula (V);

[0083] In some embodiments, the oligonucleotide can comprise a 2'-5'- phosphodiester linkage.

[0084] In some embodiments, the oligonucleotide comprises a nucleotide selected from the group consisting of: uridine-2'-phosphate (U-2'5') as shown in Formula (VI), guanosine-2'-phosphate (G-2'5') as shown in Formula (VII); cytidine-2'-phosphate (C-2'5') as shown in Formula (VIII); adenosine-2'-phosphate (A-2'5') as shown in Formula (IX), and thymidine-2'-phosphate (T-2'5') as shown in Formula (X);

[0085] In some embodiments, the oligonucleotide can comprise a glycol nucleic acid (GNA) modification.

[0086] In some embodiments, the glycol nucleic acid (GNA) modification is selected from the group consisting of: adenosine-glycol nucleic acid, cytidine-glycol nucleic acid, thymidine-glycol nucleic acid, and guanosine-glycol nucleic acid.

[0087] In some embodiments, the glycol nucleic acid (GNA) modification is selected from the group consisting of: thymidine-glycol nucleic acid S-isomer (Tgn), cytidine-glycol nucleic acid S-isomer (Cgn), adenosine-glycol nucleic acid S-isomer (Agn), and guanosine-glycol nucleic acid S-isomer (Ggn);

[0088] Structural formulas of Tgn, Cgn, Agn, and Ggn are as follows:

[0089] In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage.

[0090] In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. The phosphorothioate internucleotide linkage modification can occur at any nucleotide on either the sense strand, the antisense strand, or both strands of the chain. For example, the internucleotide linkage modification can occur at every nucleotide on either the sense strand or the antisense strand; every internucleotide linkage modification can occur in an alternating pattern on either the sense strand or the antisense strand; or either the sense strand or the antisense strand can contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand can be the same or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand can have an offset relative to the alternating pattern of internucleotide linkages on the antisense strand. In one embodiment, the double stranded RNAi agent includes from 6 to 8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand includes two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand includes at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0091] In some embodiments, the sense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 5, 6, 7, 9, 10, 11, 12, 15, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 32, 38, 68, 69, 70, 71, 72, 76, 77, 78, 82, 86, 88, 90, 96, 97, 99, 112, 128, 130, 132, 140, 146, 150, 151, 152, 154, and 165, or a modified oligonucleotide of any one of SEQ ID NO. 339, 340, 341, 343, 344, 345, 346, 349, 351, 352, 353, 354, 355, 356, 357, 358, 360, 361, 366, 371, 372, 402, 403, 404, 405, 406, 410, 411, 412, 416, 420, 422, 424, 426, 427, 431, 433, 434, 435, 437, 438, 442, 450, 453, 466, 482, 484, 494, 504, 505, 506, 508, 519, 522, 523, 525, 526, 527, 528, 530, 531, 532, 533, 535, 739, 740, 741, 742, 743, 744, 745, 746, 749, 750, 751, 752, 753, 754, 755, and 756; and the antisense strand comprises an unmodified oligonucleotide selected from any one of SEQ ID NO. 172, 173, 174, 176, 177, 178, 179, 182, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 199, 204, 205, 235, 236, 237, 238, 239, 243, 244, 245, 249, 253, 255, 257, 263, 266, 279, 295, 297, 299, 307, 313, 317, 318, 319, 321, and 332, or a modified oligonucleotide of any one of SEQ ID NO.541, 542, 543, 545, 546, 547, 548, 551, 553, 554, 555, 556, 557, 558, 559, 560, 562, 563, 568, 573, 574, 604, 605, 606, 607, 608, 612, 613, 614, 618, 622, 624, 626, 628, 629, 633, 635, 636, 637, 639, 640, 644, 652, 655, 668, 684, 686, 696, 706, 707, 708, 710, 721, 724, 725, 727, 728, 729, 730, 732, 733, 734, 735, 737, 757, 758, 759, 760, 761, 762, 763, 764, 767, 768, 769, 770, 771, 772, 773, and 774.

[0092] In some embodiments, the oligonucleotide comprises any one of the following combinations of sense and antisense strands:

[0093] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 5, and the antisense strand comprises the sequence set forth in SEQ ID NO. 172;

[0094] (2) the sense strand comprises the sequence set forth in SEQ ID NO. 6, and the antisense strand comprises the sequence set forth in SEQ ID NO. 173;

[0095] (3) the sense strand comprises the sequence set forth in SEQ ID NO. 7, and the antisense strand comprises the sequence set forth in SEQ ID NO. 174;

[0096] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 9, and the antisense strand comprises the sequence set forth in SEQ ID NO. 176;

[0097] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 10, and the antisense strand comprises the sequence set forth in SEQ ID NO. 177;

[0098] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 11, and the antisense strand comprises the sequence set forth in SEQ ID NO. 178;

[0099] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 12, and the antisense strand comprises the sequence set forth in SEQ ID NO. 179;

[0100] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 15, and the antisense strand comprises the sequence set forth in SEQ ID NO. 182;

[0101] (9) the sense strand comprises the sequence set forth in SEQ ID NO. 17, and the antisense strand comprises the sequence set forth in SEQ ID NO. 184;

[0102] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 18, and the antisense strand comprises the sequence set forth in SEQ ID NO. 185;

[0103] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 19, and the antisense strand comprises the sequence set forth in SEQ ID NO. 186;

[0104] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 20, and the antisense strand comprises the sequence set forth in SEQ ID NO. 187;

[0105] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 21, and the antisense strand comprises the sequence set forth in SEQ ID NO. 188;

[0106] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 22, and the antisense strand comprises the sequence set forth in SEQ ID NO. 189;

[0107] (15) the sense strand comprises the sequence set forth in SEQ ID NO. 23, and the antisense strand comprises the sequence set forth in SEQ ID NO. 190;

[0108] (16) the sense strand comprises the sequence set forth in SEQ ID NO. 24, and the antisense strand comprises the sequence set forth in SEQ ID NO. 191;

[0109] (17) the sense strand comprises the sequence set forth in SEQ ID NO. 26, and the antisense strand comprises the sequence set forth in SEQ ID NO. 193;

[0110] (18) the sense strand comprises the sequence set forth in SEQ ID NO. 27, and the antisense strand comprises the sequence set forth in SEQ ID NO. 194;

[0111] (19) the sense strand comprises the sequence set forth in SEQ ID NO. 32, and the antisense strand comprises the sequence set forth in SEQ ID NO. 199;

[0112] (20) the sense strand comprises the sequence set forth in SEQ ID NO. 38, and the antisense strand comprises the sequence set forth in SEQ ID NO. 205;

[0113] (21) the sense strand comprises the sequence set forth in SEQ ID NO. 68, and the antisense strand comprises the sequence set forth in SEQ ID NO. 235;

[0114] (22) the sense strand comprises the sequence set forth in SEQ ID NO. 69, and the antisense strand comprises the sequence set forth in SEQ ID NO. 236;

[0115] (23) the sense strand comprises the sequence set forth in SEQ ID NO. 70, and the antisense strand comprises the sequence set forth in SEQ ID NO. 237;

[0116] (24) the sense strand comprises the sequence set forth in SEQ ID NO. 71, and the antisense strand comprises the sequence set forth in SEQ ID NO. 238;

[0117] (25) the sense strand comprises the sequence set forth in SEQ ID NO. 72, and the antisense strand comprises the sequence set forth in SEQ ID NO. 239;

[0118] (26) the sense strand comprises the sequence set forth in SEQ ID NO. 76, and the antisense strand comprises the sequence set forth in SEQ ID NO. 243;

[0119] (27) the sense strand comprises the sequence set forth in SEQ ID NO. 77, and the antisense strand comprises the sequence set forth in SEQ ID NO. 244;

[0120] (28) the sense strand comprises the sequence set forth in SEQ ID NO. 78, and the antisense strand comprises the sequence set forth in SEQ ID NO. 245;

[0121] (29) the sense strand comprises the sequence set forth in SEQ ID NO. 82, and the antisense strand comprises the sequence set forth in SEQ ID NO. 249;

[0122] (30) the sense strand comprises the sequence set forth in SEQ ID NO. 86, and the antisense strand comprises the sequence set forth in SEQ ID NO. 253;

[0123] (31) the sense strand comprises the sequence set forth in SEQ ID NO. 88, and the antisense strand comprises the sequence set forth in SEQ ID NO. 255;

[0124] (32) the sense strand comprises the sequence set forth in SEQ ID NO. 90, and the antisense strand comprises the sequence set forth in SEQ ID NO. 257;

[0125] (33) the sense strand comprises the sequence set forth in SEQ ID NO. 96, and the antisense strand comprises the sequence set forth in SEQ ID NO. 263;

[0126] (34) the sense strand comprises the sequence set forth in SEQ ID NO. 97, and the antisense strand comprises the sequence set forth in SEQ ID NO. 235;

[0127] (35) the sense strand comprises the sequence set forth in SEQ ID NO. 99, and the antisense strand comprises the sequence set forth in SEQ ID NO. 266;

[0128] (36) the sense strand comprises the sequence set forth in SEQ ID NO. 112, and the antisense strand comprises the sequence set forth in SEQ ID NO. 279;

[0129] (37) the sense strand comprises the sequence set forth in SEQ ID NO. 128, and the antisense strand comprises the sequence set forth in SEQ ID NO. 295;

[0130] (38) the sense strand comprises the sequence set forth in SEQ ID NO. 130, and the antisense strand comprises the sequence set forth in SEQ ID NO. 297;

[0131] (39) the sense strand comprises the sequence set forth in SEQ ID NO. 140, and the antisense strand comprises the sequence set forth in SEQ ID NO. 307;

[0132] (40) the sense strand comprises the sequence set forth in SEQ ID NO. 146, and the antisense strand comprises the sequence set forth in SEQ ID NO. 313;

[0133] (41) the sense strand comprises the sequence set forth in SEQ ID NO. 150, and the antisense strand comprises the sequence set forth in SEQ ID NO. 317;

[0134] (42) the sense strand comprises the sequence set forth in SEQ ID NO. 151, and the antisense strand comprises the sequence set forth in SEQ ID NO. 318;

[0135] (43) the sense strand comprises the sequence set forth in SEQ ID NO. 152, and the antisense strand comprises the sequence set forth in SEQ ID NO. 319;

[0136] (44) the sense strand comprises the sequence set forth in SEQ ID NO. 154, and the antisense strand comprises the sequence set forth in SEQ ID NO. 321; and

[0137] (45) the sense strand comprises the sequence set forth in SEQ ID NO. 165, and the antisense strand comprises the sequence set forth in SEQ ID NO. 332.

[0138] In some embodiments, the oligonucleotide comprises any one selected from the following combinations of sense and antisense strands:

[0139] (1) the sense strand comprises a sequence set forth in SEQ ID NO. 5, and the antisense strand comprises a sequence set forth in SEQ ID NO. 172;

[0140] (2) the sense strand comprises a sequence set forth in SEQ ID NO. 6, and the antisense strand comprises a sequence set forth in SEQ ID NO. 173;

[0141] (3) the sense strand comprises a sequence set forth in SEQ ID NO. 20, and the antisense strand comprises a sequence set forth in SEQ ID NO. 187;

[0142] (4) the sense strand comprises a sequence set forth in SEQ ID NO. 32, and the antisense strand comprises a sequence set forth in SEQ ID NO. 199;

[0143] (5) the sense strand comprises a sequence set forth in SEQ ID NO. 38, and the antisense strand comprises a sequence set forth in SEQ ID NO. 205;

[0144] (6) the sense strand comprises a sequence set forth in SEQ ID NO. 69, and the antisense strand comprises a sequence set forth in SEQ ID NO. 236;

[0145] (7) the sense strand comprises a sequence set forth in SEQ ID NO. 70, and the antisense strand comprises a sequence set forth in SEQ ID NO. 237;

[0146] (8) the sense strand comprises a sequence set forth in SEQ ID NO. 78, and the antisense strand comprises a sequence set forth in SEQ ID NO. 245;

[0147] (9) the sense strand comprises a sequence set forth in SEQ ID NO. 96, and the antisense strand comprises a sequence set forth in SEQ ID NO. 263;

[0148] (10) the sense strand comprises a sequence set forth in SEQ ID NO. 97, and the antisense strand comprises a sequence set forth in SEQ ID NO. 235;

[0149] (11) the sense strand comprises a sequence set forth in SEQ ID NO. 99, and the antisense strand comprises a sequence set forth in SEQ ID NO. 266;

[0150] (12) the sense strand comprises a sequence set forth in SEQ ID NO. 112, and the antisense strand comprises a sequence set forth in SEQ ID NO. 279;

[0151] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 128, and the antisense strand comprises the sequence set forth in SEQ ID NO. 295;

[0152] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 130, and the antisense strand comprises the sequence set forth in SEQ ID NO. 297;

[0153] (15) the sense strand comprises the sequence set forth in SEQ ID NO. 140, and the antisense strand comprises the sequence set forth in SEQ ID NO. 307;

[0154] (16) the sense strand comprises the sequence set forth in SEQ ID NO. 146, and the antisense strand comprises the sequence set forth in SEQ ID NO. 313;

[0155] (17) the sense strand comprises the sequence set forth in SEQ ID NO. 150, and the antisense strand comprises the sequence set forth in SEQ ID NO. 317;

[0156] (18) the sense strand comprises the sequence set forth in SEQ ID NO. 151, and the antisense strand comprises the sequence set forth in SEQ ID NO. 318;

[0157] (19) the sense strand comprises the sequence set forth in SEQ ID NO. 152, and the antisense strand comprises the sequence set forth in SEQ ID NO. 319;

[0158] (20) the sense strand comprises the sequence set forth in SEQ ID NO. 154, and the antisense strand comprises the sequence set forth in SEQ ID NO. 321; and

[0159] (21) the sense strand comprises the sequence set forth in SEQ ID NO. 165, and the antisense strand comprises the sequence set forth in SEQ ID NO. 332.

[0160] In some embodiments, the oligonucleotide comprises any one of the sense and antisense strand combinations selected from:

[0161] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 6, and the antisense strand comprises the sequence set forth in SEQ ID NO. 173;

[0162] (2) the sense strand comprises the sequence set forth in SEQ ID NO. 20, and the antisense strand comprises the sequence set forth in SEQ ID NO. 187;

[0163] (3) the sense strand comprises the sequence set forth in SEQ ID NO. 32, and the antisense strand comprises the sequence set forth in SEQ ID NO. 199;

[0164] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 38, and the antisense strand comprises the sequence set forth in SEQ ID NO. 205;

[0165] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 70, and the antisense strand comprises the sequence set forth in SEQ ID NO. 237;

[0166] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 96, and the antisense strand comprises the sequence set forth in SEQ ID NO. 263;

[0167] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 99, and the antisense strand comprises the sequence set forth in SEQ ID NO. 266;

[0168] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 128, and the antisense strand comprises the sequence set forth in SEQ ID NO. 295;

[0169] (9) the sense strand comprises the sequence set forth in SEQ ID NO. 130, and the antisense strand comprises the sequence set forth in SEQ ID NO. 297;

[0170] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 150, and the antisense strand comprises the sequence set forth in SEQ ID NO. 317; and

[0171] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 165, and the antisense strand comprises the sequence set forth in SEQ ID NO. 332.

[0172] In some embodiments, the oligonucleotide comprises any one of the sense and antisense strand combinations selected from:

[0173] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 339, and the antisense strand comprises the sequence set forth in SEQ ID NO. 541;

[0174] (2) the sense strand comprises the sequence set forth in SEQ ID NO. 340, and the antisense strand comprises the sequence set forth in SEQ ID NO. 542;

[0175] (3) the sense strand comprises the sequence set forth in SEQ ID NO. 341, and the antisense strand comprises the sequence set forth in SEQ ID NO. 543;

[0176] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 343, and the antisense strand comprises the sequence set forth in SEQ ID NO. 545;

[0177] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 344, and the antisense strand comprises the sequence set forth in SEQ ID NO. 546;

[0178] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 345, and the antisense strand comprises the sequence set forth in SEQ ID NO. 547;

[0179] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 346, and the antisense strand comprises the sequence set forth in SEQ ID NO. 548;

[0180] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 349, and the antisense strand comprises the sequence set forth in SEQ ID NO. 551;

[0181] (9) the sense strand comprises the sequence set forth in SEQ ID NO. 351, and the antisense strand comprises the sequence set forth in SEQ ID NO. 553;

[0182] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 352, and the antisense strand comprises the sequence set forth in SEQ ID NO. 554;

[0183] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 353, and the antisense strand comprises the sequence set forth in SEQ ID NO. 555;

[0184] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 354, and the antisense strand comprises the sequence set forth in SEQ ID NO. 556;

[0185] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 355, and the antisense strand comprises the sequence set forth in SEQ ID NO. 557;

[0186] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 356, and the antisense strand comprises the sequence set forth in SEQ ID NO. 558;

[0187] (15) the sense strand comprises the sequence set forth in SEQ ID NO. 357, and the antisense strand comprises the sequence set forth in SEQ ID NO. 559;

[0188] (16) the sense strand comprises the sequence set forth in SEQ ID NO. 358, and the antisense strand comprises the sequence set forth in SEQ ID NO. 560;

[0189] (17) the sense strand comprises the sequence set forth in SEQ ID NO. 360, and the antisense strand comprises the sequence set forth in SEQ ID NO. 562;

[0190] (18) the sense strand comprises the sequence set forth in SEQ ID NO. 361, and the antisense strand comprises the sequence set forth in SEQ ID NO. 563;

[0191] (19) the sense strand comprises the sequence set forth in SEQ ID NO. 366, and the antisense strand comprises the sequence set forth in SEQ ID NO. 568;

[0192] (20) the sense strand comprises the sequence set forth in SEQ ID NO. 371, and the antisense strand comprises the sequence set forth in SEQ ID NO. 573;

[0193] (21) the sense strand comprises the sequence set forth in SEQ ID NO. 372, and the antisense strand comprises the sequence set forth in SEQ ID NO. 574;

[0194] (22) the sense strand comprises the sequence set forth in SEQ ID NO. 402, and the antisense strand comprises the sequence set forth in SEQ ID NO. 604;

[0195] (23) the sense strand comprises the sequence set forth in SEQ ID NO. 403, and the antisense strand comprises the sequence set forth in SEQ ID NO. 605;

[0196] (24) the sense strand comprises the sequence set forth in SEQ ID NO. 404, and the antisense strand comprises the sequence set forth in SEQ ID NO. 606;

[0197] (25) the sense strand comprises the sequence set forth in SEQ ID NO. 405, and the antisense strand comprises the sequence set forth in SEQ ID NO. 607;

[0198] (26) the sense strand comprises the sequence set forth in SEQ ID NO. 406, and the antisense strand comprises the sequence set forth in SEQ ID NO. 608;

[0199] (27) the sense strand comprises the sequence set forth in SEQ ID NO. 410, and the antisense strand comprises the sequence set forth in SEQ ID NO. 612;

[0200] (28) the sense strand comprises the sequence set forth in SEQ ID NO. 411, and the antisense strand comprises the sequence set forth in SEQ ID NO. 613;

[0201] (29) the sense strand comprises the sequence set forth in SEQ ID NO. 412, and the antisense strand comprises the sequence set forth in SEQ ID NO. 614;

[0202] (30) the sense strand comprises the sequence set forth in SEQ ID NO. 416, and the antisense strand comprises the sequence set forth in SEQ ID NO. 618;

[0203] (31) the sense strand comprises the sequence set forth in SEQ ID NO. 420, and the antisense strand comprises the sequence set forth in SEQ ID NO. 622;

[0204] (32) the sense strand comprises the sequence set forth in SEQ ID NO. 422, and the antisense strand comprises the sequence set forth in SEQ ID NO. 624;

[0205] (33) the sense strand comprises the sequence set forth in SEQ ID NO. 424, and the antisense strand comprises the sequence set forth in SEQ ID NO. 626;

[0206] (34) the sense strand comprises the sequence set forth in SEQ ID NO. 426, and the antisense strand comprises the sequence set forth in SEQ ID NO. 628;

[0207] (35) the sense strand comprises the sequence set forth in SEQ ID NO. 427, and the antisense strand comprises the sequence set forth in SEQ ID NO. 629;

[0208] (36) the sense strand comprises the sequence set forth in SEQ ID NO. 431, and the antisense strand comprises the sequence set forth in SEQ ID NO. 633;

[0209] (37) the sense strand comprises the sequence set forth in SEQ ID NO. 434, and the antisense strand comprises the sequence set forth in SEQ ID NO. 636;

[0210] (38) the sense strand comprises the sequence set forth in SEQ ID NO. 435, and the antisense strand comprises the sequence set forth in SEQ ID NO. 637;

[0211] (39) the sense strand comprises the sequence set forth in SEQ ID NO. 437, and the antisense strand comprises the sequence set forth in SEQ ID NO. 639;

[0212] (40) the sense strand comprises the sequence set forth in SEQ ID NO. 438, and the antisense strand comprises the sequence set forth in SEQ ID NO. 640;

[0213] (41) the sense strand comprises the sequence set forth in SEQ ID NO. 442, and the antisense strand comprises the sequence set forth in SEQ ID NO. 644;

[0214] (42) the sense strand comprises the sequence set forth in SEQ ID NO. 450, and the antisense strand comprises the sequence set forth in SEQ ID NO. 652;

[0215] (43) the sense strand comprises the sequence set forth in SEQ ID NO. 453, and the antisense strand comprises the sequence set forth in SEQ ID NO. 655;

[0216] (44) the sense strand comprises the sequence set forth in SEQ ID NO. 466, and the antisense strand comprises the sequence set forth in SEQ ID NO. 668;

[0217] (45) the sense strand comprises the sequence set forth in SEQ ID NO. 482, and the antisense strand comprises the sequence set forth in SEQ ID NO. 684;

[0218] (46) the sense strand comprises the sequence set forth in SEQ ID NO. 484, and the antisense strand comprises the sequence set forth in SEQ ID NO. 686;

[0219] (47) the sense strand comprises the sequence set forth in SEQ ID NO. 494, and the antisense strand comprises the sequence set forth in SEQ ID NO. 696;

[0220] (48) the sense strand comprises the sequence set forth in SEQ ID NO. 504, and the antisense strand comprises the sequence set forth in SEQ ID NO. 706;

[0221] (49) the sense strand comprises the sequence set forth in SEQ ID NO. 505, and the antisense strand comprises the sequence set forth in SEQ ID NO. 707;

[0222] (50) the sense strand comprises the sequence set forth in SEQ ID NO. 506, and the antisense strand comprises the sequence set forth in SEQ ID NO. 708;

[0223] (51) the sense strand comprises the sequence set forth in SEQ ID NO. 508, and the antisense strand comprises the sequence set forth in SEQ ID NO. 710;

[0224] (52) the sense strand comprises the sequence set forth in SEQ ID NO. 519, and the antisense strand comprises the sequence set forth in SEQ ID NO. 721;

[0225] (53) the sense strand comprises the sequence set forth in SEQ ID NO. 522, and the antisense strand comprises the sequence set forth in SEQ ID NO. 724;

[0226] (54) the sense strand comprises the sequence set forth in SEQ ID NO. 523, and the antisense strand comprises the sequence set forth in SEQ ID NO. 725;

[0227] (55) the sense strand comprises the sequence set forth in SEQ ID NO. 524, and the antisense strand comprises the sequence set forth in SEQ ID NO. 726;

[0228] (56) the sense strand comprises the sequence set forth in SEQ ID NO. 525, and the antisense strand comprises the sequence set forth in SEQ ID NO. 727;

[0229] (57) the sense strand comprises the sequence set forth in SEQ ID NO. 526, and the antisense strand comprises the sequence set forth in SEQ ID NO. 728;

[0230] (58) the sense strand comprises the sequence set forth in SEQ ID NO. 527, and the antisense strand comprises the sequence set forth in SEQ ID NO. 729;

[0231] (59) the sense strand comprises the sequence set forth in SEQ ID NO. 530, and the antisense strand comprises the sequence set forth in SEQ ID NO. 732;

[0232] (60) the sense strand comprises the sequence set forth in SEQ ID NO. 531, and the antisense strand comprises the sequence set forth in SEQ ID NO. 733;

[0233] (61) the sense strand comprises the sequence set forth in SEQ ID NO. 532, and the antisense strand comprises the sequence set forth in SEQ ID NO. 734;

[0234] (62) the sense strand comprises the sequence set forth in SEQ ID NO. 533, and the antisense strand comprises the sequence set forth in SEQ ID NO. 735;

[0235] (63) the sense strand comprises the sequence set forth in SEQ ID NO. 535, and the antisense strand comprises the sequence set forth in SEQ ID NO. 737;

[0236] (64) the sense strand comprises the sequence set forth in SEQ ID NO. 433, and the antisense strand comprises the sequence set forth in SEQ ID NO. 635;

[0237] (65) the sense strand comprises the sequence set forth in SEQ ID NO. 739, and the antisense strand comprises the sequence set forth in SEQ ID NO. 757;

[0238] (66) the sense strand comprises the sequence set forth in SEQ ID NO. 740, and the antisense strand comprises the sequence set forth in SEQ ID NO. 758;

[0239] (67) the sense strand comprises the sequence set forth in SEQ ID NO. 741, and the antisense strand comprises the sequence set forth in SEQ ID NO. 759;

[0240] (68) the sense strand comprises the sequence set forth in SEQ ID NO. 742, and the antisense strand comprises the sequence set forth in SEQ ID NO. 760;

[0241] (69) the sense strand comprises the sequence set forth in SEQ ID NO. 743, and the antisense strand comprises the sequence set forth in SEQ ID NO. 761;

[0242] (70) the sense strand comprises the sequence set forth in SEQ ID NO. 744, and the antisense strand comprises the sequence set forth in SEQ ID NO. 762;

[0243] (71) the sense strand comprises the sequence set forth in SEQ ID NO. 745, and the antisense strand comprises the sequence set forth in SEQ ID NO. 763;

[0244] (72) the sense strand comprises the sequence set forth in SEQ ID NO. 746, and the antisense strand comprises the sequence set forth in SEQ ID NO. 764;

[0245] (73) the sense strand comprises the sequence set forth in SEQ ID NO. 749, and the antisense strand comprises the sequence set forth in SEQ ID NO. 767;

[0246] (74) the sense strand comprises the sequence set forth in SEQ ID NO. 750, and the antisense strand comprises the sequence set forth in SEQ ID NO. 768;

[0247] (75) the sense strand comprises the sequence set forth in SEQ ID NO. 751, and the antisense strand comprises the sequence set forth in SEQ ID NO. 769;

[0248] (76) the sense strand comprises the sequence set forth in SEQ ID NO. 752, and the antisense strand comprises the sequence set forth in SEQ ID NO. 770;

[0249] (77) the sense strand comprises the sequence set forth in SEQ ID NO. 753, and the antisense strand comprises the sequence set forth in SEQ ID NO. 771;

[0250] (78) the sense strand comprises the sequence set forth in SEQ ID NO. 754, and the antisense strand comprises the sequence set forth in SEQ ID NO. 772;

[0251] (79) the sense strand comprises the sequence set forth in SEQ ID NO. 755, and the antisense strand comprises the sequence set forth in SEQ ID NO. 773; and

[0252] (80) the sense strand comprises the sequence set forth in SEQ ID NO. 756, and the antisense strand comprises the sequence set forth in SEQ ID NO. 774.

[0253] In some embodiments, the oligonucleotide comprises any one of the following combinations of sense and antisense strands:

[0254] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 354, and the antisense strand comprises the sequence set forth in SEQ ID NO. 556;

[0255] (2) the sense strand comprises the sequence set forth in SEQ ID NO. 371, and the antisense strand comprises the sequence set forth in SEQ ID NO. 509;

[0256] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 426, and the antisense strand comprises the sequence set forth in SEQ ID NO. 628;

[0257] (3) the sense strand comprises the sequence set forth in SEQ ID NO. 427, and the antisense strand comprises the sequence set forth in SEQ ID NO. 629;

[0258] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 431, and the antisense strand comprises the sequence set forth in SEQ ID NO. 633;

[0259] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 434, and the antisense strand comprises the sequence set forth in SEQ ID NO. 636;

[0260] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 435, and the antisense strand comprises the sequence set forth in SEQ ID NO. 637;

[0261] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 437, and the antisense strand comprises the sequence set forth in SEQ ID NO. 639;

[0262] (9) the sense strand comprises the sequence set forth in SEQ ID NO. 438, and the antisense strand comprises the sequence set forth in SEQ ID NO. 640;

[0263] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 442, and the antisense strand comprises the sequence set forth in SEQ ID NO. 644;

[0264] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 450, and the antisense strand comprises the sequence set forth in SEQ ID NO. 652;

[0265] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 453, and the antisense strand comprises the sequence set forth in SEQ ID NO. 655;

[0266] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 466, and the antisense strand comprises the sequence set forth in SEQ ID NO. 668;

[0267] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 482, and the antisense strand comprises the sequence set forth in SEQ ID NO. 684;

[0268] (15) the sense strand comprises the sequence set forth in SEQ ID NO. 484, and the antisense strand comprises the sequence set forth in SEQ ID NO. 686;

[0269] (16) the sense strand comprises the sequence set forth in SEQ ID NO. 494, and the antisense strand comprises the sequence set forth in SEQ ID NO. 696;

[0270] (17) the sense strand comprises the sequence set forth in SEQ ID NO. 504, and the antisense strand comprises the sequence set forth in SEQ ID NO. 706;

[0271] (18) the sense strand comprises the sequence set forth in SEQ ID NO. 505, and the antisense strand comprises the sequence set forth in SEQ ID NO. 707;

[0272] (19) the sense strand comprises the sequence set forth in SEQ ID NO. 506, and the antisense strand comprises the sequence set forth in SEQ ID NO. 708;

[0273] (20) the sense strand comprises the sequence set forth in SEQ ID NO. 508, and the antisense strand comprises the sequence set forth in SEQ ID NO. 710;

[0274] (21) the sense strand comprises the sequence set forth in SEQ ID NO. 519, and the antisense strand comprises the sequence set forth in SEQ ID NO. 721;

[0275] (22) the sense strand comprises the sequence set forth in SEQ ID NO. 522, and the antisense strand comprises the sequence set forth in SEQ ID NO. 724;

[0276] (23) the sense strand comprises the sequence set forth in SEQ ID NO. 523, and the antisense strand comprises the sequence set forth in SEQ ID NO. 725;

[0277] (24) the sense strand comprises the sequence set forth in SEQ ID NO. 526, and the antisense strand comprises the sequence set forth in SEQ ID NO. 728;

[0278] (25) the sense strand comprises the sequence set forth in SEQ ID NO. 527, and the antisense strand comprises the sequence set forth in SEQ ID NO. 729;

[0279] (26) the sense strand comprises the sequence set forth in SEQ ID NO. 532, and the antisense strand comprises the sequence set forth in SEQ ID NO. 734;

[0280] (27) the sense strand comprises the sequence set forth in SEQ ID NO. 535, and the antisense strand comprises the sequence set forth in SEQ ID NO. 737;

[0281] (28) the sense strand comprises the sequence set forth in SEQ ID NO. 433, and the antisense strand comprises the sequence set forth in SEQ ID NO. 635;

[0282] (29) the sense strand comprises the sequence set forth in SEQ ID NO. 525, and the antisense strand comprises the sequence set forth in SEQ ID NO. 727;

[0283] (30) the sense strand comprises the sequence set forth in SEQ ID NO. 533, and the antisense strand comprises the sequence set forth in SEQ ID NO. 735;

[0284] (31) the sense strand comprises the sequence set forth in SEQ ID NO. 739, and the antisense strand comprises the sequence set forth in SEQ ID NO. 757;

[0285] (32) the sense strand comprises the sequence set forth in SEQ ID NO. 740, and the antisense strand comprises the sequence set forth in SEQ ID NO. 758;

[0286] (33) the sense strand comprises the sequence set forth in SEQ ID NO. 741, and the antisense strand comprises the sequence set forth in SEQ ID NO. 759;

[0287] (34) the sense strand comprises the sequence set forth in SEQ ID NO. 742, and the antisense strand comprises the sequence set forth in SEQ ID NO. 760;

[0288] (35) the sense strand comprises the sequence set forth in SEQ ID NO. 743, and the antisense strand comprises the sequence set forth in SEQ ID NO. 761;

[0289] (36) the sense strand comprises the sequence set forth in SEQ ID NO. 744, and the antisense strand comprises the sequence set forth in SEQ ID NO. 762;

[0290] (37) the sense strand comprises the sequence set forth in SEQ ID NO. 745, and the antisense strand comprises the sequence set forth in SEQ ID NO. 763;

[0291] (38) the sense strand comprises the sequence set forth in SEQ ID NO. 746, and the antisense strand comprises the sequence set forth in SEQ ID NO. 764;

[0292] (39) the sense strand comprises the sequence set forth in SEQ ID NO. 749, and the antisense strand comprises the sequence set forth in SEQ ID NO. 767;

[0293] (40) the sense strand comprises the sequence set forth in SEQ ID NO. 750, and the antisense strand comprises the sequence set forth in SEQ ID NO. 768;

[0294] (41) the sense strand comprises the sequence set forth in SEQ ID NO. 751, and the antisense strand comprises the sequence set forth in SEQ ID NO. 769;

[0295] (42) the sense strand comprises the sequence set forth in SEQ ID NO. 752, and the antisense strand comprises the sequence set forth in SEQ ID NO. 770;

[0296] (43) the sense strand comprises the sequence set forth in SEQ ID NO. 753, and the antisense strand comprises the sequence set forth in SEQ ID NO. 771;

[0297] (44) the sense strand comprises the sequence set forth in SEQ ID NO. 754, and the antisense strand comprises the sequence set forth in SEQ ID NO. 772;

[0298] (45) the sense strand comprises the sequence set forth in SEQ ID NO. 755, and the antisense strand comprises the sequence set forth in SEQ ID NO. 773; and

[0299] (46) the sense strand comprises the sequence set forth in SEQ ID NO. 756, and the antisense strand comprises the sequence set forth in SEQ ID NO. 774.

[0300] In some embodiments, the oligonucleotide comprises any one of the sense and antisense strand combinations selected from:

[0301] (1) the sense strand comprises the sequence set forth in SEQ ID NO. 354, and the antisense strand comprises the sequence set forth in SEQ ID NO. 556;

[0302] (2) the sense strand comprises the sequence set forth in SEQ ID NO. 427, and the antisense strand comprises the sequence set forth in SEQ ID NO. 629;

[0303] (3) the sense strand comprises the sequence set forth in SEQ ID NO. 431, and the antisense strand comprises the sequence set forth in SEQ ID NO. 633;

[0304] (4) the sense strand comprises the sequence set forth in SEQ ID NO. 434, and the antisense strand comprises the sequence set forth in SEQ ID NO. 636;

[0305] (5) the sense strand comprises the sequence set forth in SEQ ID NO. 435, and the antisense strand comprises the sequence set forth in SEQ ID NO. 637;

[0306] (6) the sense strand comprises the sequence set forth in SEQ ID NO. 438, and the antisense strand comprises the sequence set forth in SEQ ID NO. 640;

[0307] (7) the sense strand comprises the sequence set forth in SEQ ID NO. 522, and the antisense strand comprises the sequence set forth in SEQ ID NO. 724;

[0308] (8) the sense strand comprises the sequence set forth in SEQ ID NO. 523, and the antisense strand comprises the sequence set forth in SEQ ID NO. 725;

[0309] (9) the sense strand comprises the sequence set forth in SEQ ID NO. 526, and the antisense strand comprises the sequence set forth in SEQ ID NO. 728;

[0310] (10) the sense strand comprises the sequence set forth in SEQ ID NO. 527, and the antisense strand comprises the sequence set forth in SEQ ID NO. 729;

[0311] (11) the sense strand comprises the sequence set forth in SEQ ID NO. 532, and the antisense strand comprises the sequence set forth in SEQ ID NO. 734;

[0312] (12) the sense strand comprises the sequence set forth in SEQ ID NO. 535, and the antisense strand comprises the sequence set forth in SEQ ID NO. 737;

[0313] (13) the sense strand comprises the sequence set forth in SEQ ID NO. 433, and the antisense strand comprises the sequence set forth in SEQ ID NO. 635;

[0314] (14) the sense strand comprises the sequence set forth in SEQ ID NO. 442, and the antisense strand comprises the sequence set forth in SEQ ID NO. 644;

[0315] (15) the sense strand comprises the sequence set forth in SEQ ID NO. 525, and the antisense strand comprises the sequence set forth in SEQ ID NO. 727; and

[0316] (16) the sense strand comprises the sequence set forth in SEQ ID NO. 533, and the antisense strand comprises the sequence set forth in SEQ ID NO. 735.

[0317] In another aspect, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting expression of INHBE, comprising: (i) the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

[0318] In some embodiments, at least one nucleotide of the foregoing oligonucleotide or salt thereof is conjugated to one or more targeting ligands to form an siRNA conjugate. The foregoing siRNA conjugate contains an siRNA described above and a conjugate group conjugated to the siRNA. The term "oligonucleotide salt" refers to a salt form of an oligonucleotide compound. Oligonucleotide salts include salts of oligonucleotide conjugate compounds as well as salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in solid powder form.

[0319] Generally, the foregoing conjugate group comprises a pharmaceutically acceptable at least one targeting ligand and optionally a linker, and the foregoing siRNA, the foregoing linker, and the foregoing targeting ligand are sequentially linked. The targeting group can be a ligand conventionally used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, the foregoing targeting ligand is 2-4. The foregoing siRNA molecule can be conjugated to the foregoing conjugate group non-covalently or covalently, for example, can be covalently conjugated to the foregoing conjugate group. The conjugation site of the siRNA to the conjugate group can be at the 3' end or 5' end of the sense strand or the antisense strand of the siRNA, and can also be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the foregoing siRNA to the conjugate group is at the 3' terminal end or 5' terminal end of the sense strand of the siRNA. In some embodiments, the conjugation site of the foregoing siRNA to the conjugate group is at the 3' terminal end or 5' terminal end of the antisense strand of the siRNA. In some preferred embodiments, the conjugation site of the foregoing siRNA to the conjugate group is at the 3' terminal end of the sense strand of the siRNA.

[0320] In some embodiments, the targeting ligand comprises an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives having an affinity for the asialoglycoprotein receptor that is equal to or greater than the affinity of galactose. Galactose derivatives include, but are not limited to, galactose, galactosamine, N-formyl galactosamine, N-acetyl galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyryl galactosamine. Galactose derivatives and clusters of galactose derivatives that can be used to target oligonucleotides and other molecules to the liver in vivo are known in the art. Galactose derivatives have been used to target molecules to hepatocytes in vivo by their binding to asialoglycoprotein receptors (ASGPrs) expressed on the surface of hepatocytes. Binding of ASGPr ligands to ASGPr(s) facilitates cell-specific targeting of hepatocytes and endocytosis of molecules into hepatocytes. ASGPr ligands can be monomeric (e.g., having a single galactose derivative) or multimeric (e.g., having multiple galactose derivatives). Galactose derivatives or clusters of galactose derivatives can be linked to the 3' end or 5' end of an siRNA using methods known in the art.

[0321] In some embodiments, the pharmaceutically acceptable targeting ligand in the foregoing siRNA conjugates can be galactose or N-acetylgalactosamine (GalNAc), wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, tetravalent. It should be understood that the monovalent, divalent, trivalent, tetravalent refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate formed after the siRNA molecule is conjugated to the conjugation group containing the galactose or N-acetylgalactosamine molecule as the targeting ligand is 1:1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharmaceutically acceptable targeting ligand is N-acetylgalactosamine. In some embodiments, when the siRNA described herein is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0322] In some embodiments, the targeting ligand is selected from a carbohydrate, an aminosugar, a cholesterol, a polypeptide, or a lipid.

[0323] In some embodiments, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0324] In some embodiments, the foregoing GalNAc moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0325] In some embodiments, the targeting ligand is L96;

[0326] In another aspect, the disclosure provides a composition comprising the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0327] In some embodiments, the dosage form of the composition is an oral dosage, an intravenous injection, a subcutaneous injection, or an intramuscular injection, preferably a subcutaneous injection.

[0328] In some embodiments, the composition further comprises another therapeutic or / and prophylactic agent for INHBE-associated disorders.

[0329] In another aspect, the disclosure provides the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or conjugate or a composition for use in the manufacture of a medicament for the treatment or / and prevention of INHBE-associated disorders.

[0330] In some embodiments, the INHBE-associated diseases, disorders, and / or conditions are selected from the group consisting of liver diseases (e.g., fatty liver, steatohepatitis), dyslipidemia (e.g., hyperlipidemia, high LDL-C, low HDL-C, hypertriglyceridemia, postprandial hypertriglyceridemia), impaired glycemic control (e.g., insulin resistance, diabetes), cardiovascular diseases (e.g., hypertension, endothelial cell dysfunction), kidney diseases (e.g., acute kidney disorders, proximal tubule dysfunction, proinflammatory changes in the proximal tubule), metabolic syndrome, adipocyte dysfunction, visceral fat deposition, obesity, hyperuricemia, gout, eating disorders, and excessive craving for sugar; and combinations thereof.

[0331] In some embodiments of the methods of the disclosure, the expression of the INHBE gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In preferred embodiments, the expression of INHBE is inhibited by at least 70%. It will also be appreciated that it can be desirable to inhibit the expression of INHBE in certain tissues (e.g., liver) without significantly inhibiting expression in other tissues (e.g., brain). In preferred embodiments, the level of expression is determined using the assay method provided in Example 2 at siRNA concentrations of 50 nM, 10 nM, and 1 nM in a suitable species-matched cell line.

[0332] In some embodiments, inhibition of expression in vivo is determined by knockdown of the human gene in a rodent expressing the human gene, for example, an AAV infected mouse expressing a human target gene (i.e., INHBE), for example, the effect of inhibition of the human gene is confirmed by the nadir of INHBE expression after, for example, a single dose administration, for example, a 3 mg / kg subcutaneous injection. Such systems are useful when the nucleic acid sequence of the human gene and the model animal gene are sufficiently close such that a human iRNA provides effective knockdown of the model animal gene. RNA expression in the liver is determined using the PCR method provided in Example 2.

[0333] Inhibition of INHBE gene expression can be represented by a decrease in the amount of mRNA expressed by a cell line (such cells can be present in, for example, a sample derived from a subject), the INHBE gene is transcribed in the cell or cell population and it is treated (for example, by contacting one or more cells with an iRNA of the disclosure, or by administering an iRNA of the disclosure to a subject in which the cells are present or were once present), such that expression of the INHBE gene is inhibited as compared to a cell line that is substantially identical to the cell line but is not treated (control cells that are not treated with an iRNA or are not treated with an iRNA targeting the gene of interest). In preferred embodiments, inhibition is assessed in a species-matched cell line using 1 nM and or 10 nM siRNA concentrations by the method provided in Example 2, using the following formula, expressed as the amount of mRNA expression in the treated cells relative to the mRNA level in the control cells: CT INHBE - CT GAPDH CT 处理细胞 - CT 对照细胞 mRNA level = 2^-△△CT

[0334] In other embodiments, inhibition of INHBE gene expression can be assessed according to a decrease in a parameter related to the function of INHBE gene expression, for example, INHBE protein levels in blood or serum from a subject. INHBE gene silencing can be determined in any cell expressing INHBE, whether endogenous or heterologous from an expression construct, and by any assay known in the art.

[0335] Inhibition of INHBE protein expression can be manifested by a decrease in the level of INHBE protein expressed by a cell or cell population or in a sample from a subject (e.g., protein levels in a blood sample from a subject). As described above, to assess mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can similarly be expressed as a percentage of protein levels in control cells or cell populations, or changes in protein levels in a sample from a subject (e.g., blood or serum therefrom). Percent inhibition of mRNA = (protein expression in treated cells 处理细胞 - protein expression in control cells 对照细胞 / protein expression in control cells 对照细胞 * 100%

[0336] Control cells, cell populations, or samples from a subject that can be used to assess inhibition of INHBE gene expression include cells, cell populations, or samples from a subject that have not been contacted with an RNAi agent of the disclosure. For example, control cells, cell lines, or samples from a subject can be from an individual subject (e.g., a human or animal subject) prior to treatment of the subject or appropriately matched population control with an RNAi agent.

[0337] In some embodiments of the methods of the disclosure, an iRNA is administered to a subject such that the iRNA is delivered to a specific site within the subject. Inhibition of INHBE expression can be assessed by measuring the level or change in INHBE mRNA or INHBE protein or fusion secreted luciferase in a sample of fluid or tissue from a specific site in the subject (e.g., the liver or blood), or by detecting biomarkers of blood glucose, glycated hemoglobin (HbAlc), blood glucose, total cholesterol, etc. to assess the effect of the iRNA.

[0338] The present disclosure also provides methods of using an iRNA of the present disclosure or a composition comprising an iRNA of the present disclosure to inhibit INHBE expression, thereby preventing or treating diseases, disorders, and / or conditions including, but not limited to, obesity, type II diabetes, hyperlipidemia or dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid moieties), obesity (particularly abdominal obesity), lipodystrophy (such as an inability to deposit fat locally in fat depots (partial lipodystrophy) or an inability to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenge, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis, or liver inflammation), nonalcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, higher or elevated or altered other markers of inflammation or fat deposition in the liver, higher and / or hypertension, higher and / or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the above conditions.

[0339] Various formulations have been developed to facilitate the use of oligonucleotides. For example, an oligonucleotide can be delivered to a subject or cellular environment using a formulation that minimizes degradation, facilitates delivery and / or uptake, or provides another beneficial property to the oligonucleotide in the formulation. In some embodiments, provided herein are compositions comprising an oligonucleotide (e.g., a single- or double-stranded oligonucleotide) to reduce expression of INHBE. Such compositions can be suitably formulated such that, when administered to a subject (either directly into the environment of the target cells or systemically), a sufficient portion of the oligonucleotide enters the cells to reduce INHBE expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver an oligonucleotide for reducing INHBE as disclosed herein. In some embodiments, the oligonucleotide or conjugate thereof is formulated in a buffered solution, such as a phosphate buffered saline solution, a liposome, a micellar structure, and a capsid. The buffered solution can be selected from a solution of acetate, citrate, glutelin, carbonate, or phosphate, or any combination thereof. In some embodiments, the naked (i.e., without a delivery agent) oligonucleotide or conjugate thereof is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the naked oligonucleotide or conjugate thereof is formulated in a basic buffered aqueous solution (e.g., PBS).

[0340] Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the oligonucleotide into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used.

[0341] Accordingly, in some embodiments, the formulation comprises a lipid nanoparticle. In some embodiments, the excipient comprises a liposome, a lipid, a lipoplex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or can otherwise be formulated for administration to a cell, tissue, organ, or body of a subject in need thereof.

[0342] In some embodiments, the formulation as disclosed herein comprises an excipient. In some embodiments, the excipient imparts to the composition increased stability, increased absorption, increased solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized for extended shelf life, and then made into a solution prior to use (e.g., administration to a subject). Accordingly, the excipient in a composition comprising any of the oligonucleotides described herein can be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0343] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

[0344] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The foregoing carriers can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotide in the required amount in the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0345] In some embodiments, the composition can contain at least about 0.1% or more of the therapeutic agent (e.g., an oligonucleotide for reducing INHBE expression), although the percentage of active ingredient(s) can be between about 1% and about 80% or more by weight or volume of the total composition. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, and other pharmacological considerations, and therefore a wide variety of dosages and treatment regimes can be desirable.

[0346] Cells suitable for treatment using the methods of the disclosure can be any cell that expresses an INHBE gene, for example, a hepatocyte, a brain cell, or a kidney cell, but preferably a hepatocyte. Cells suitable for use in the methods of the disclosure can be mammalian cells, for example, primate cells (such as human cells, including human cells in chimeric non-human animals, or non-human primate cells, for example, monkey cells or chimpanzee cells) or non-primate cells. In some embodiments, the cells are human cells, for example, human hepatocytes. In the methods of the disclosure, expression of INHBE in the cells is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection of the assay.

[0347] The in vivo methods of the disclosure can include administering to a subject a composition comprising an iRNA, wherein the iRNA comprises a nucleotide sequence that is complementary to at least a portion of an RNA transcript of an INHBE gene of the mammal to which the RNAi agent is administered. The composition can be administered by any means known in the art, including, but not limited to, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered intramuscularly.

[0348] In one aspect, the present disclosure also provides a method for inhibiting expression of an INHBE gene in a mammal. The method comprises administering to the mammal an oligonucleotide or a pharmaceutically acceptable salt thereof or a composition thereof. The oligonucleotide is a double-stranded RNA (dsRNA) that targets the INHBE gene in a mammalian cell and is maintained in the mammal for a sufficient time to obtain degradation of the mRNA transcript of the INHBE gene, thereby inhibiting expression of the INHBE protein in the cell. Reduction in gene expression can be assessed by any method known in the art and by methods, e.g., qRT-PCR, described herein, e.g., in Example 2. Reduction in the protein product can be assessed by any method known in the art, e.g., ELISA. In other embodiments, blood samples are taken as subject samples for monitoring reduction in INHBE protein expression.

[0349] The present disclosure also provides a method of treatment in a subject in need thereof, e.g., a subject diagnosed with an INHBE-related disorder, including but not limited to obesity, type II diabetes, hyperlipidemia or dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid fractions), obesity (particularly abdominal obesity), lipodystrophy (such as an inability to deposit fat locally in fat depots (partial lipodystrophy) or an inability to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenge, liver fat deposition or fatty liver disease and its complications (such as, e.g., cirrhosis, fibrosis, or liver inflammation), nonalcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, other markers of inflammation or fat deposition in the liver higher or elevated or altered, higher and / or high blood pressure, higher and / or high blood glucose, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the above conditions.

[0350] In one embodiment, INHBE-related diseases include, but are not limited to, obesity, type II diabetes, hyperlipidemia or dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid fractions), obesity (particularly abdominal obesity), lipodystrophy (such as an inability to deposit fat locally in fat depots (partial lipodystrophy) or an inability to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenge, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis, or liver inflammation), nonalcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, other markers of inflammation or fat deposition in the liver higher or elevated or altered, higher and / or high blood pressure, higher or high blood glucose or blood sugar, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the above conditions.

[0351] The iRNAs of the present disclosure can be administered as “free iRNAs.” Free iRNAs are administered without a pharmaceutical composition. Naked iRNAs can be in a suitable buffered solution. The buffered solution can comprise acetate, citrate, lactate, tartrate, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffered solution containing the iRNA can be adjusted to be suitable for administration to a subject.

[0352] Administration of the iRNAs according to the methods of the present disclosure can result in the prevention or treatment of INHBE-related conditions, including, but not limited to, obesity, type II diabetes, hyperlipidemia or dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid fractions), obesity (particularly abdominal obesity), lipodystrophy (such as an inability to deposit fat locally in fat depots (partial lipodystrophy) or an inability to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenge, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis, or liver inflammation), nonalcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, other markers of inflammation or fat deposition in the liver higher or elevated or altered, higher and / or high blood pressure, higher or high blood glucose or blood sugar, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the above conditions.

[0353] A therapeutic amount of iRNA can be administered to a subject, such as a dose in the range of about 0.01 mg / kg to about 200 mg / kg (e.g., about 0.1 mg / kg to about 100 mg / kg). In some embodiments, the oligonucleotide is administered to a subject in a range of about 0.1 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 20 mg / kg, 0.3 mg / kg to about 18 mg / kg, 0.5 mg / kg to about 15 mg / kg, or 0.5 mg / kg to about 12 mg / kg, more preferably about 1 mg / kg to about 10 mg / kg. Preferably 1 mg / kg to about 50 mg / kg. The iRNA is preferably administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver the desired dose of iRNA to a subject. Injections can be repeated over a period of time.

[0354] In some embodiments, the iRNA is administered at a fixed dose of about 50 mg to about 800 mg. In some embodiments, the iRNA is administered to a subject at a fixed dose of about 50 mg to about 200 mg, about 200 mg to about 400 mg, or about 400 mg to about 800 mg. In some embodiments, the iRNA is administered to a subject at a fixed dose of about about 100 mg, about 200 mg, about 300 mg, about 400 mg, 500 mg, about 600 mg, about 700 mg, or about 800 mg.

[0355] The administration can be repeated periodically. In some embodiments, after an initial treatment regimen, treatment can be performed at a lower frequency. The repeated dosing regimen can include periodic administration of a therapeutic amount of iRNA, such as from once a month to once a year. In some embodiments, the iRNA administration is about once a month to about once every three months, or about once every three months to about once every six months, or even once a year.

[0356] In some embodiments, the fixed dose is administered to the subject at an interval of once a month. In some embodiments, the fixed dose is administered to the subject at an interval of once every six months.

[0357] In some embodiments, the subject is administered a fixed dose of about 150 mg once about every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg once about every six months. In some embodiments, the subject is administered a fixed dose of about 300 mg once about every six months. In some embodiments, the subject is administered a fixed dose of about 600 mg once about every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg once about every six months. In some embodiments, the subject is administered a fixed dose of about 800 mg once about every six months.

[0358] In some embodiments, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammalian subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats and chickens; and laboratory animals such as mice, rats, guinea pigs, and hamsters.

[0359] The present disclosure further provides that the iRNA agent or pharmaceutical composition thereof in combination with other drugs and / or other therapeutic methods (e.g., known drugs and / or known therapeutic methods, e.g., those currently used to treat these conditions) will benefit patients with liver diseases, dyslipidemia, cardiovascular diseases, impaired glycemic control, etc. The combination drugs can be oral drugs including: metformin, alpha-glucosidase inhibitors, insulin secretagogues (sulfonylureas and glinides), thiazolidinediones (TZDs), DPP4 inhibitors, SGLT2 inhibitors. Injection drugs include: GLP1 receptor agonists and insulin. Lipid-lowering drugs include statins, fibrates, cholesterol absorption inhibitors, proprotein convertase subtilisin 9 (PCSK9) inhibitors, etc. Drugs that inhibit uric acid synthesis, such as xanthine oxidase inhibitors (XOIs): XOIs inhibit uric acid synthesis, including allopurinol and febuxostat; drugs that increase uric acid excretion, represented by benzbromarone and probenecid, etc.

[0360] For the purpose of clarity and brevity, some features are described herein as part of the same or separate embodiments, however, it is to be understood that the scope of the present disclosure can include embodiments having combinations of all or some of the described features.

[0361] Hereinafter, the present disclosure will be described in more detail with reference to specific examples, however, the examples are for illustrative purposes only and do not limit the present disclosure.

[0362] Examples

[0363] Example 1. Preparation of targeting ligands and siRNAs

[0364] In cases where the source of reagents is not specified herein, such reagents can be obtained from any molecular biology reagent supplier, which quality / purity standards are suitable for molecular biology.

[0365] Abbreviations used for nucleotide monomers in nucleic acid sequence representation. It is understood that when a nucleotide contains a 2'-fluoro modification, then the fluorine replaces the hydroxyl group at that position in the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluoro nucleotide).

[0366] Table A. Abbreviations for nucleotide monomers used in nucleic acid sequence representation

[0367] Preparation of targeting ligands

[0368] Preparation of L96 was performed according to the method described in patent CN104717982B.

[0369] Preparation of oligonucleotides

[0370] (1) Preparation of siRNA

[0371] First, computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human INHBE mRNA (NM_006488.3, Table 1), some of which are also complementary to cynomolgus monkey INHBE mRNA (NM_031479.5, Table 1) or no more than 2 mismatches. Some of them are designed as double-stranded siRNA with 19 / 21 pairing of sense and antisense strands respectively, the antisense strand with two overhanging ends complementary to mRNA sequence, in some cases the overhanging ends of the antisense strand are non-complementary UU; some of them are designed as double-stranded siRNA with 21 / 23 pairing of sense and antisense strands respectively, the antisense strand with two overhanging ends complementary to mRNA sequence; some of them are designed as double-stranded siRNA with 21 / 21, 23 / 23 pairing. The 1st base at the 5' end of the antisense strand of some complementary pairing sequences (the last 1st base at the 3' end of the sense strand) is replaced with a base that does not match INHBE mRNA.

[0372] Table 1 Human, cynomolgus monkey INHBE mRNA sequence

[0373] The siRNA sequences were synthesized separately on a solid support via the sense strand (SS) and the antisense strand (AS), and after deprotection, cleavage, purification, annealing, purification and freeze-drying, the siRNA sequences were obtained.

[0374] Solid phase synthesis (Figure 4): The sense strand and the antisense strand were synthesized separately on a solid support using the oligonucleotide synthesizer by phosphoramidite technique. The synthesizer was AKTA Oligopilot (Cytiva), Dr. Oligo 192XLc (Kunshan Boly Precision Instrument Co., Ltd.). The solid phase synthesis started from the 3' end of the sequence, and the monomers were coupled into the sequence in sequence order. Each coupling of a phosphoramidite monomer included four chemical steps: 1) deprotection or deprotection of the hydroxyl protecting group; 2) coupling; 3) oxidation; 4) capping. The phosphoramidite monomers, reagents, and purification consumables used were commercialized and circulated reagents and consumables, such as various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zilve Science and Technology Development Co., Ltd., and reaction reagents (such as 40wt% methylamine aqueous solution, 28wt% ammonium hydroxide aqueous solution, etc.) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this paper are described in US20130178612A1, US2015100197A1, etc.; the synthesis method of the sequence containing VPUm and APU structure is described in J. Med. Chem. 2018, 61, 734-744.

[0375] (2) Preparation of double-stranded RNA (dsRNA) agents

[0376] (a) Synthesis of the sense strand

[0377] Solid phase phosphoramidite method is a mature oligonucleotide synthesis method. A computer-controlled synthesizer is used to perform the reaction in a stainless steel synthesis column. In the synthesis of the sense strand, a solid support loaded with a targeting ligand (such as L96) is used as the starting material, or a solid support is directly used as the starting material. Different raw materials, reagents, and solvents are injected into different pipelines in sequence 3' to 5' order under the control of the solid phase synthesizer, and phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four steps of deprotection of DMT protecting group reaction, condensation reaction, oxidation or thio reaction, and capping reaction. Each cycle connects one nucleotide unit, and an oligonucleotide sequence of 19 or 21 nucleotide units is obtained. After synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid phase synthesis column, and the synthesized sequence is then cut from the solid support through an aminolysis reaction. Filtration is performed, the filter cake is washed with ethanol, and the filtrate and washing liquid are collected and concentrated to obtain the crude sense strand. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to obtain the target product, the sense strand. In the synthesizer, a solid support loaded with a targeting ligand (such as L96) is used as the starting material to synthesize the siRNA sense strand conjugate; a solid support is directly used as the starting material to synthesize the siRNA.

[0378] (b) synthesis of antisense strand

[0379] The synthesis of antisense strand is similar to that of sense strand. The different raw materials, reagents and solvents are injected into the different pipes according to the sequence 3' to 5' order to connect the phosphoramidite nucleoside monomers one by one under the control of a solid-phase synthesizer. The reaction process includes four steps of cycles, i.e. removal of DMT protecting group reaction, condensation reaction, oxidation or thio reaction, and capping reaction, one nucleotide unit is connected each cycle to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After the synthesis is completed, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and the synthesized sequence is then cut from the solid-phase support through an ammonolysis reaction, filtered, washed with ethanol, and the filtrate and washing liquid are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltration, and lyophilization to obtain the target product antisense strand siRNA.

[0380] (c) preparation of double-stranded siRNA

[0381] The AS strand and the SS strand are dissolved in injection water respectively, mixed in a certain ratio (1.01:1.0-1.2:1.0), incubated at 30-50°C for 30-90 min, and cooled to room temperature. After freeze-drying, the double-stranded siRNA product is obtained.

[0382] According to the same method, the double-stranded siRNA agents in Tables 2, 3 and 4 below are prepared.

[0383] In Tables 2, 3 and 4, "G", "C", "A", "U" and "T" generally represent nucleotides with guanine, cytosine, adenine, uracil and thymine as the base, respectively. The naked sequence in Tables 2, 3 and 4 refers to an unmodified oligonucleotide sequence.

[0384] Modification: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluorine; s represents phosphorothioate; VPUm is 2'-methoxy modified uridine; L96 is N-[tris(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3); dA represents 2'-deoxyadenosine-3'-phosphate; dG represents 2'-deoxyguanosine-3'-phosphate; dC represents 2'-deoxycytidine-3'-phosphate; dU represents 2'-deoxyuridine-3'-phosphate.

[0385] Certain oligonucleotides can comprise glycol nucleic acid (GNA) modifications. In certain oligonucleotides, individual ribonucleotides are replaced with (Tgn), which is a thymidine-glycol nucleic acid (GNA) S-isomer of Formula (I). In certain oligonucleotides, individual ribonucleotides are replaced with (Cgn), which is a cytidine-glycol nucleic acid (GNA) S-isomer of Formula (II). In certain oligonucleotides, individual ribonucleotides are replaced with (Agn), which is an adenosine-glycol nucleic acid (GNA) S-isomer of Formula (III). In certain oligonucleotides, individual ribonucleotides are replaced with (Ggn), which is a guanosine-glycol nucleic acid (GNA) S-isomer of Formula (IV). The structural formulas of Tgn, Cgn, Agn, and Ggn are as follows:

[0386] Table 2. Oligonucleotide naked sequences

[0387] Table 3. Oligonucleotide modified sequences

[0388] Table 4. Oligonucleotide modified sequences

[0389] Example 2. In vitro activity evaluation of INHBE-siRNA in cell lines

[0390] (1) Cell culture and transfection:

[0391] Hep3B cells (Wuhan Ponsay Life Science Co., Ltd., Cat. No. CL-0102) were placed in a 37°C, 5% CO2 incubator, cultured with DMEM medium (Sibeliawell Biotech Co., Ltd., Shanghai, Cat. No. iCell-0001) added with 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122). When the cell confluence reached 90%, the cells were digested with trypsin-EDTA (Thermo, 25200-072), counted with a counting instrument (Countstar, IC1000), and 190 μl of cell suspension was inoculated per well in a 96-well plate. The inoculation number of Hep3B cells was 2*104 Cells / well, to be adherent next day for transfection.

[0392] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150), 2.2 μl (2 μM) diluted compound, 19.1 μl Opti-MEM (thermofisher, 1105821), 0.7 μl RNAiMAX were mixed to form transfection complex, incubated for 5 min, then the transfection complex was added to cells (two technical replicates per complex), 10 μl per well, the final concentration of siRNA was 10 nM, incubated at 37℃, 5% CO2 incubator for 24 hours.

[0393] (2) RNA extraction and detection

[0394] (i) Total RNA was extracted using RNA-Quick Purification Kit (Yishan Biotech, RN001):

[0395] The 12-well plate was taken out of the incubator, the medium was aspirated, washed once with appropriate PBS, 500 μl of lysis buffer was added to each well, and the supernatant was transferred to a new 1.5 ml centrifuge tube. 500 μl of absolute ethanol was added to the lysed cells and mixed well (if a precipitate appears, it is a normal phenomenon, continue the operation). The centrifuge tube was inverted several times, or the precipitate was dispersed by blowing and sucking with a pipette for 10 times, then the liquid was added to the centrifugal column, and the centrifuge tube was placed symmetrically in the centrifuge (eppendorf, 5430) at 4000 x g for 1 min. The centrifuge tube was taken out and 500 μl of wash buffer was added to the column, which was centrifuged at 12000 x g for 1 min. After centrifugation, the column was taken out and the waste liquid was discarded. The column was placed in a clean RNAse-free 1.5 ml centrifuge tube, and the cap was opened to dry for 2 minutes. 30 μl of elution buffer was added to the center of the RNA column membrane, and it was left to stand at room temperature for 2 minutes, and then centrifuged at 2000 x g for 1 min. After the RNA was eluted, it was placed on ice. The concentration of eluted RNA was determined for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments, or stored at -80℃ for later use.

[0396] (ii) cDNA was synthesized using IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novozyme, R223-01):

[0397] Prepare the mixture in a RNase-free centrifuge tube: 4 μl 4×gDNA wiper Mix, 1 μg template RNA, add RNase-free ddH2O to 16 μl to remove genomic DNA, mix gently with a pipette, 42℃ for 2 min. Then add 4 μl 5×HiScript II qRT SuperMix II directly to the reaction tube, mix gently with a pipette, and place in a PCR instrument (Applied Biosystems, 9700) at 50℃ for 15 min, 85℃ for 5 sec, and 4℃ for holding. The product can be used immediately for qPCR reaction, or stored at -20℃ and used within half a year, or stored at -80℃ after sub-packaging for long-term storage. The cDNA should be avoided from repeated freezing and thawing.

[0398] (iii) Quantification by qPCR using ChamQ SYBR qPCR Master Mix (Norgen, Q311-02):

[0399] Prepare the mixture: 10 μl 2×ChamQ SYBR qPCR Master Mix, 0.5 μl Forword primer (Ribobio), 0.5 μl Reverse primer (Ribobio), 1 μl Template cDNA, 8 μl ddH2O to 20 μl system, 3 repeats for each sample, and place the 96-well plate in a qPCR instrument (ROCGENE, Archimed) to execute the program: pre-denaturation, 95℃ for 30 sec; amplification, 95℃ for 10 sec, 60℃ for 30 sec, 40 cycles; melting curve, 95℃ for 15 sec, 60℃ for 60 sec, 95℃ for 15 sec.

[0400] (3) Statistical analysis of data:

[0401] Export the data to EXCEL format, and calculate CT INHBE -CT GAPDH Normalize the control group, and calculate the fold change of relative silencing efficiency by using the △△CT method to analyze the data, and calculate the average value and standard deviation of the obtained three parallel repeat data.

[0402] The screening results of Hep3B cells in two times are shown in Tables 4-6. The data in each table are obtained from a single test, and the silencing efficiency of target genes will be different due to different batches of cells.

[0403] As shown in Table 4, at a dosage of 10 nM, the inhibition rate of INHBE mRNA of some siRNAs can reach 50% or more, such as AL0245005, AL0245006, AL0245007, AL0245009, AL0245010, AL0245011, AL0245012, AL0245015, AL0245017, AL0245018, AL0245019, AL0245020, AL0245021, AL0245023, AL0245024, AL0245026, AL0245027, AL0245032, AL0245038, AL0245068, AL0245069, AL0245070, AL0245071, AL0245072, AL0245076, AL0245077, AL0245078, AL0245082, AL0245086, AL0245088 and AL0245090; the inhibition rate of INHBE mRNA of some sequences can reach more than 80%, such as AL0245020. To further increase the efficacy of the sequences, the preferred sequences with an inhibition rate of about 60% are added with VPUm modification, and the dose efficacy evaluation at dosages of 10 nM and 1 nM is performed, and the detection results are shown in Table 5. As shown in the results, when the VPUm modification is added, compared with AL0245001, the efficacy of most sequences is further improved, such as AL0245092, AL0245097, AL0245099, AL0245100, AL0245101, AL0245103, AL0245104 and AL0245108, and at a dosage of 1 nM, the inhibition rate of the target gene mRNA can reach or be close to 70%.

[0404] In another set of siRNA target gene mRNA silencing efficiency evaluation experiments, to more effectively evaluate the silencing effect of different siRNAs, the dosage is further reduced to 0.5 nM, and the experimental results are shown in Table 6. As shown in the results, at a dosage of 0.5 nM, the efficacy of some sequences can still reach 30% or more, such as AL0245116, AL0245119, AL0245132, AL0245148, AL0245150, AL0245160, AL0245170, AL0245171, AL0245172, AL0245174 and AL0245185.

[0405] Table 5 INHBE siRNA knockdown level in Hep3B

[0406] Table 6 INHBE siRNA knockdown level in Hep3B

[0407] Table 7 INHBE siRNA knockdown levels in Hep3B

[0408] Example 3. INHBE-siRNA in vitro activity evaluation in monkey primary hepatocytes

[0409] (1) Cell culture and transfection:

[0410] Cynomolgus monkey hepatocytes (Beijing Ruidebaio Biological Technology Co., Ltd., cmTCSC) were used. The culture medium was preheated, and the thawing medium (Beijing Ruidebaio Biological Technology Co., Ltd., HEPO24) was taken out of the biosafety cabinet, 4 mL of FBS was added to 36 mL of thawing medium (TPCS, HEPO24) to make complete thawing medium, and heated in a 37°C water bath for 10 minutes. The coating medium (Beijing Ruidebaio Biological Technology Co., Ltd., HEPO44) was treated at 37°C in a CO2 incubator for 0.5 h. The cells were taken out from liquid nitrogen, and the cells were recovered in a 37°C water bath for about 2 min, then taken out, and the cell suspension was transferred to the preheated 40 mL thawing medium, and the cell cryopreservation tube was washed with 2 mL of complete thawing medium, and the cell suspension was centrifuged at 180 x g for 1 min, and the supernatant was discarded, 2 mL of preheated CM seeding medium (Beijing Ruidebaio Biological Technology Co., Ltd., CMHEP054) was added, and the cell suspension was gently blown and mixed, and 20 μl of cell suspension was counted. According to the counting result, 12-well plates were inoculated, 3*10 5 cells / well, and placed in a 37°C, 5% CO2 incubator for culture. After 4-5 h of adhesion, the CM seeding medium was aspirated and replaced with preheated culture medium (Beijing Ruidebaio Biological Technology Co., Ltd., CMHEP064), and transfection was performed after 6 h of adhesion.

[0411] Lipofectamine TM RNAiMAX (thermofisher, 13778150) was used for transfection, 2.2 μl (1 μM or 200 mM or 100 nM or 20 mM) diluted compound, 19.1 μl Opti-MEM (thermofisher, 1105821), 0.7 μl RNAiMAX were mixed to form a transfection complex, and incubated for 5 min, then the transfection complex was added to the cells (two technical repeats per complex), 10 μl per well, the final concentration of siRNA was 5 nM or 1 nM or 0.5 or 0.1, and incubated at 37°C, 5% CO2 incubator for 24 h.

[0412] (2) RNA extraction and detection

[0413] The same as Example 2.

[0414] (3) Data statistical analysis:

[0415] The same as Example 2.

[0416] As can be seen from Table 7 and Table 8, in cynomolgus monkey primary hepatocytes, when the dosage is 1 nM and 0.1 nM, the inhibition rate of all sequences on INHBE mRNA can reach more than 70%, except for AL0245092, AL0245095, AL0245096, AL0245103, AL0245191, AL0245195, AL0245196, AL0245197 and AL0245200, especially AL0245020, AL0245093, AL0245097, AL0245100, AL0245101, AL0245104, AL0245188, AL0245189, AL0245192, AL0245193, AL0245198 and AL0245201 sequences, the inhibition rate of INHBE mRNA can reach 80% or close to 80% at a dosage of 0.1 nM, and the inhibition rate of target gene mRNA reaches or is close to 90% at a dosage of 1 nM.

[0417] Table 8 INHBE siRNA knockdown level in cynomolgus monkey primary hepatocytes

[0418] Table 9 INHBE siRNA knockdown level in cynomolgus monkey primary hepatocytes

[0419] As shown in Table 10 and Table 12, in cynomolgus monkey primary hepatocytes, when the dosage is 5 nM and 0.5 nM, the inhibition rates of AL0245097, AL0245100, AL0245101, AL0245188, AL0245192, AL0245199, AL0245201, AL0245203, AL0245204, AL0245205, AL0245206, AL0245207, AL0245208, AL0245209, AL0245210, AL0245213, AL0245214, AL0245215, AL0245216, AL0245217, AL0245218, AL0245219, AL0245220 on INHBE mRNA can all reach more than 70%, and in particular, the inhibition rates of AL0245097, AL0245101, AL0245188, AL0245192, AL0245201, AL0245203, AL0245204, AL0245205, AL0245206, AL0245208, AL0245209, AL0245210, AL0245213, AL0245214, AL0245216 on INHBE mRNA can all reach more than 80%; the inhibition rates of AL0245188, AL0245192, AL0245201, AL0245209, AL0245210, AL0245213 on INHBE mRNA can all reach more than 90%.

[0420] As shown in Table 11, in cynomolgus monkey primary hepatocytes, when the dosage is 5 nM, except for AL0247003, the inhibition rates of AL0247001, AL0247002, AL0247004, AL0247005, AL0247006, AL0247007, AL0247008, AL0247009, AL0247010, AL0247011, AL0247012, AL0247013, AL0247014 on INHBE mRNA can all reach more than 60%.

[0421] Table 10 INHBE siRNA knockdown level in cynomolgus monkey primary hepatocytes

[0422] Table 11 INHBE siRNA knockdown level in cynomolgus monkey primary hepatocytes

[0423] Table 12 INHBE siRNA knockdown level in cynomolgus monkey primary hepatocytes

[0424] Example 4. In vivo testing of INHBE RNAi agents in transgenic mice

[0425] SPF level male, 8-9 weeks old C57BL / 6J / hINHBE mice (Nanjing Biomedical) were used in the experiment. The mice were given a single subcutaneous administration of 3 mg / kg of RNAi agent, and a control group (NC) was set as the solvent group, which was given the same volume of 0.9% saline. On day 22 after administration, the knockdown level of hINHBE expression in liver tissue was detected with the saline group (solvent group) as the control. The extraction and detection of mRNA and data analysis were performed according to Example 3. During the experiment, no death or near-death symptoms were observed in all animals. No obvious abnormalities were observed in clinical observation of all animals. The changes in the expression level of hINHBE mRNA are shown in Figure 2. According to the experimental results, the silencing effect of AL0247009, AL0247010, AL0247013 and AL0247014 on the target gene 22 days after administration can reach about 50% or even lower compared with the NC group.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting INHBE expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having a sequence with at least 60% or more sequence identity to the sequence set forth in any one of SEQ ID NO. 3-167 or a fragment thereof or a modified sequence thereof, preferably having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity; the antisense strand having a sequence with at least 60% or more sequence identity to the sequence set forth in any one of SEQ ID NO. 170-334 or a fragment thereof or a modified sequence thereof, preferably having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity.

2. The oligonucleotide or pharmaceutically acceptable salt thereof of claim 1, wherein, the pharmaceutically acceptable salt of the oligonucleotide is selected from the group consisting of a carboxylate salt, an alkali metal salt, an ammonium salt, an alkaline earth metal salt, a salt with an organic base, and other pharmaceutically acceptable salts; preferably, the salt is an alkali metal salt, more preferably a sodium salt or a potassium salt; preferably, the salt is an alkaline earth metal salt, more preferably a magnesium salt or a calcium salt; preferably, the salt is an ammonium salt.

3. The oligonucleotide or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein, the oligonucleotide comprises at least one modified nucleotide; preferably, the at least one modified nucleotide in the modified nucleotide is selected from the group consisting of a deoxynucleotide, a 3' terminal deoxythymidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a 2'-5'-linked ribonucleotide (3'-RNA), an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyranyl modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate analog, a vinyl-phosphonate nucleotide, a thermally unstable nucleotide, a glycol modified nucleotide, a nucleotide comprising a 2' phosphate, and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof; preferably, the at least one modified nucleotide in the modified nucleotide is selected from the group consisting of a LNA modified nucleotide, a HNA modified nucleotide, a CeNA modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-allyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a 2'-hydroxyl modified nucleotide, and a glycol modified nucleotide; and combinations thereof; Preferably, the at least one modified nucleotide in the modified nucleotide is selected from the group consisting of a deoxynucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a glycol modified nucleotide, a nucleotide comprising a 2' phosphate, and a nucleotide comprising a phosphorothioate group; and combinations thereof; Preferably, the oligonucleotide comprises at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotide is selected from the group consisting of a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-acylamino modified nucleotide, a 2'-deoxy modified nucleotide, a 2'-0-allyl modified nucleotide, a 2'-0-alkyl modified nucleotide, a 2'-hydroxyl modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, a 2'-deoxynucleotide, a nucleotide comprising a 2' phosphate, and a 2'-0-(N-methylacetamide) modified nucleotide; and combinations thereof; Preferably, the 2'-modification is selected from the group consisting of a 2'-methoxy, a 2'-acetamido, a 2'-aminoethyl, a 2'-fluoro, a 2'-0-methyl, and a 2'-0-methoxyethyl modification; and combinations thereof; Preferably, the oligonucleotide comprises a modification at the 5' terminus, the modification comprising a 5'-phosphate analog or a 6-(3-(2-carboxyethyl)phenyl)purine (6-mCEPh-purine); Preferably, the 5'-phosphate analogue modification is a 5'-(E)-vinylphosphonate (5'-VP); preferably, the 5' terminal nucleotide comprising the 5'-(E)-vinylphosphonate modification has the structure shown in Formula (I); wherein Base represents a natural or modified base; preferably Base is selected from A, G, C and U; R is selected from H, fluoro, 2'-methoxy, 2'-acetamido, 2'-aminoethyl and 2'-0-methoxyethyl; preferably, the 5'-phosphate analogue modified nucleotide has the structure shown in Formula (II); more preferably, the 5'-phosphonate analogue modified nucleotide is an APU of Formula (III) or a VPUm of Formula (IV); Preferably, the oligonucleotide comprises a 6-(3-(2-carboxyethyl)phenyl) purine modified nucleotide at the 5' terminus; preferably, the oligonucleotide comprises Formula M, which is a 2'-0-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide as shown in Formula (V); 4. The oligonucleotide or pharmaceutically acceptable salt thereof of any one of claims 1-3, wherein, the oligonucleotide comprises at least one modified internucleotide linkage; Preferably, the at least one modified internucleotide linkage is a phosphorothioate linkage.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, the antisense strand comprises an unmodified oligonucleotide selected from any of SEQ ID NOs. 172, 173, 174, 176, 177, 178, 179, 182, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 199, 204, 205, 235, 236, 237, 238, 239, 243, 244, 245, 249, 253, 255, 257, 263, 266, 279, 295, 297, 299, 307, 313, 317, 318, 319, 321, and 332, or a modified oligonucleotide of any of SEQ ID NOs. 339, 340, 341, 343, 344, 345, 346, 349, 351, 352, 353, 354, 355, 356, 357, 358, 360, 361, 366, 371, 372, 402, 403, 404, 405, 406, 410, 411, 412, 416, 420, 422, 424, 426, 427, 431, 433, 434, 435, 437, 438, 442, 450, 453, 466, 482, 484, 494, 504, 505, 506, 508, 519, 522, 523, 525, 526, 527, 528, 530, 531, 532, 533, 535, 739, 740, 741, 742, 743, 744, 745, 746, 749, 750, 751, 752, 753, 754, 755, and 756.541, 542, 543, 545, 546, 547, 548, 551, 553, 554, 555, 556, 557, 558, 559, 560, 562, 563, 568, 573, 574, 604, 605, 606, 607, 608, 612, 613, 614, 618, 622, 624, 626, 628, 629, 633, 635, 636, 637, 639, 640, 644, 652, 655, 668, 684, 686, 696, 706, 707, 708, 710, 721, 724, 725, 727, 728, 729, 730, 732, 733, 734, 735, 737, 757, 758, 759, 760, 761, 762, 763, 764, 767, 768, 769, 770, 771, 772, 773, and 774.

6. The oligonucleotide or a pharmaceutically acceptable salt thereof of any one of claims 1-5, wherein, the oligonucleotide comprises any one of the following sense and antisense strand combinations: (1) the sense strand comprises the sequence set forth in SEQ ID NO. 5, and the antisense strand comprises the sequence set forth in SEQ ID NO. 172; (2) the sense strand comprises the sequence set forth in SEQ ID NO. 6, and the antisense strand comprises the sequence set forth in SEQ ID NO. 173; (3) the sense strand comprises the sequence set forth in SEQ ID NO. 7, and the antisense strand comprises the sequence set forth in SEQ ID NO. 174; (4) the sense strand comprises the sequence set forth in SEQ ID NO. 9, and the antisense strand comprises the sequence set forth in SEQ ID NO. 176; (5) the sense strand comprises the sequence set forth in SEQ ID NO. 10, and the antisense strand comprises the sequence set forth in SEQ ID NO. 177; (6) the sense strand comprises the sequence set forth in SEQ ID NO. 11, and the antisense strand comprises the sequence set forth in SEQ ID NO. 178; (7) the sense strand comprises the sequence set forth in SEQ ID NO. 12, and the antisense strand comprises the sequence set forth in SEQ ID NO. 179; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 15, and the antisense strand comprises a sequence set forth in SEQ ID NO. 182; (9) the sense strand comprises a sequence set forth in SEQ ID NO. 17, and the antisense strand comprises a sequence set forth in SEQ ID NO. 184; (10) the sense strand comprises a sequence set forth in SEQ ID NO. 18, and the antisense strand comprises a sequence set forth in SEQ ID NO. 185; (11) the sense strand comprises a sequence set forth in SEQ ID NO. 19, and the antisense strand comprises a sequence set forth in SEQ ID NO. 186; (12) the sense strand comprises a sequence set forth in SEQ ID NO. 20, and the antisense strand comprises a sequence set forth in SEQ ID NO. 187; (13) the sense strand comprises a sequence set forth in SEQ ID NO. 21, and the antisense strand comprises a sequence set forth in SEQ ID NO. 188; (14) the sense strand comprises a sequence set forth in SEQ ID NO. 22, and the antisense strand comprises a sequence set forth in SEQ ID NO. 189; (15) the sense strand comprises a sequence set forth in SEQ ID NO. 23, and the antisense strand comprises a sequence set forth in SEQ ID NO. 190; (16) the sense strand comprises a sequence set forth in SEQ ID NO. 24, and the antisense strand comprises a sequence set forth in SEQ ID NO. 191; (17) the sense strand comprises a sequence set forth in SEQ ID NO. 26, and the antisense strand comprises a sequence set forth in SEQ ID NO. 193; (18) the sense strand comprises a sequence set forth in SEQ ID NO. 27, and the antisense strand comprises a sequence set forth in SEQ ID NO. 194; (19) the sense strand comprises a sequence set forth in SEQ ID NO. 32, and the antisense strand comprises a sequence set forth in SEQ ID NO. 199; (20) the sense strand comprises a sequence set forth in SEQ ID NO. 38, and the antisense strand comprises a sequence set forth in SEQ ID NO. 205; (21) the sense strand comprises a sequence set forth in SEQ ID NO. 68, and the antisense strand comprises a sequence set forth in SEQ ID NO. 235; (22) the sense strand comprises a sequence set forth in SEQ ID NO. 69, and the antisense strand comprises a sequence set forth in SEQ ID NO. 236; (23) the sense strand comprises a sequence set forth in SEQ ID NO. 70, and the antisense strand comprises a sequence set forth in SEQ ID NO. 237; (24) the sense strand comprises a sequence set forth in SEQ ID NO. 71, and the antisense strand comprises a sequence set forth in SEQ ID NO. 238; (25) the sense strand comprises the sequence set forth in SEQ ID NO. 72, and the antisense strand comprises the sequence set forth in SEQ ID NO. 239; (26) the sense strand comprises the sequence set forth in SEQ ID NO. 76, and the antisense strand comprises the sequence set forth in SEQ ID NO. 243; (27) the sense strand comprises the sequence set forth in SEQ ID NO. 77, and the antisense strand comprises the sequence set forth in SEQ ID NO. 244; (28) the sense strand comprises the sequence set forth in SEQ ID NO. 78, and the antisense strand comprises the sequence set forth in SEQ ID NO. 245; (29) the sense strand comprises the sequence set forth in SEQ ID NO. 82, and the antisense strand comprises the sequence set forth in SEQ ID NO. 249; (30) the sense strand comprises the sequence set forth in SEQ ID NO. 86, and the antisense strand comprises the sequence set forth in SEQ ID NO. 253; (31) the sense strand comprises the sequence set forth in SEQ ID NO. 88, and the antisense strand comprises the sequence set forth in SEQ ID NO. 255; (32) the sense strand comprises the sequence set forth in SEQ ID NO. 90, and the antisense strand comprises the sequence set forth in SEQ ID NO. 257; (33) the sense strand comprises the sequence set forth in SEQ ID NO. 96, and the antisense strand comprises the sequence set forth in SEQ ID NO. 263; (34) the sense strand comprises the sequence set forth in SEQ ID NO. 97, and the antisense strand comprises the sequence set forth in SEQ ID NO. 235; (35) the sense strand comprises the sequence set forth in SEQ ID NO. 99, and the antisense strand comprises the sequence set forth in SEQ ID NO. 266; (36) the sense strand comprises the sequence set forth in SEQ ID NO. 112, and the antisense strand comprises the sequence set forth in SEQ ID NO. 279; (37) the sense strand comprises the sequence set forth in SEQ ID NO. 128, and the antisense strand comprises the sequence set forth in SEQ ID NO. 295; (38) the sense strand comprises the sequence set forth in SEQ ID NO. 130, and the antisense strand comprises the sequence set forth in SEQ ID NO. 297; (39) the sense strand comprises the sequence set forth in SEQ ID NO. 140, and the antisense strand comprises the sequence set forth in SEQ ID NO. 307; (40) the sense strand comprises the sequence set forth in SEQ ID NO. 146, and the antisense strand comprises the sequence set forth in SEQ ID NO. 313; (41) the sense strand comprises the sequence set forth in SEQ ID NO. 150, and the antisense strand comprises the sequence set forth in SEQ ID NO. 317; (42) the sense strand comprises a sequence set forth in SEQ ID NO. 151, and the antisense strand comprises a sequence set forth in SEQ ID NO. 318; (43) the sense strand comprises a sequence set forth in SEQ ID NO. 152, and the antisense strand comprises a sequence set forth in SEQ ID NO. 319; (44) the sense strand comprises a sequence set forth in SEQ ID NO. 154, and the antisense strand comprises a sequence set forth in SEQ ID NO. 321; and (45) the sense strand comprises a sequence set forth in SEQ ID NO. 165, and the antisense strand comprises a sequence set forth in SEQ ID NO. 332; Preferably, the oligonucleotide comprises any one of a sense strand and an antisense strand combination selected from: (1) the sense strand comprises a sequence set forth in SEQ ID NO. 5, and the antisense strand comprises a sequence set forth in SEQ ID NO. 172; (2) the sense strand comprises a sequence set forth in SEQ ID NO. 6, and the antisense strand comprises a sequence set forth in SEQ ID NO. 173; (3) the sense strand comprises a sequence set forth in SEQ ID NO. 20, and the antisense strand comprises a sequence set forth in SEQ ID NO. 187; (4) the sense strand comprises a sequence set forth in SEQ ID NO. 32, and the antisense strand comprises a sequence set forth in SEQ ID NO. 199; (5) the sense strand comprises a sequence set forth in SEQ ID NO. 38, and the antisense strand comprises a sequence set forth in SEQ ID NO. 205; (6) the sense strand comprises a sequence set forth in SEQ ID NO. 69, and the antisense strand comprises a sequence set forth in SEQ ID NO. 236; (7) the sense strand comprises a sequence set forth in SEQ ID NO. 70, and the antisense strand comprises a sequence set forth in SEQ ID NO. 237; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 78, and the antisense strand comprises a sequence set forth in SEQ ID NO. 245; (9) the sense strand comprises a sequence set forth in SEQ ID NO. 96, and the antisense strand comprises a sequence set forth in SEQ ID NO. 263; (10) the sense strand comprises a sequence set forth in SEQ ID NO. 97, and the antisense strand comprises a sequence set forth in SEQ ID NO. 235; (11) the sense strand comprises a sequence set forth in SEQ ID NO. 99, and the antisense strand comprises a sequence set forth in SEQ ID NO. 266; (12) the sense strand comprises a sequence set forth in SEQ ID NO. 112, and the antisense strand comprises a sequence set forth in SEQ ID NO. 279; (13) the sense strand comprises a sequence set forth in SEQ ID NO. 128, and the antisense strand comprises a sequence set forth in SEQ ID NO. 295; (14) the sense strand comprises a sequence set forth in SEQ ID NO. 130, and the antisense strand comprises a sequence set forth in SEQ ID NO. 297; (15) the sense strand comprises a sequence set forth in SEQ ID NO. 140, and the antisense strand comprises a sequence set forth in SEQ ID NO. 307; (16) the sense strand comprises a sequence set forth in SEQ ID NO. 146, and the antisense strand comprises a sequence set forth in SEQ ID NO. 313; (17) the sense strand comprises a sequence set forth in SEQ ID NO. 150, and the antisense strand comprises a sequence set forth in SEQ ID NO. 317; (18) the sense strand comprises a sequence set forth in SEQ ID NO. 151, and the antisense strand comprises a sequence set forth in SEQ ID NO. 318; (19) the sense strand comprises a sequence set forth in SEQ ID NO. 152, and the antisense strand comprises a sequence set forth in SEQ ID NO. 319; (20) the sense strand comprises a sequence set forth in SEQ ID NO. 154, and the antisense strand comprises a sequence set forth in SEQ ID NO. 321; and (21) the sense strand comprises a sequence set forth in SEQ ID NO. 165, and the antisense strand comprises a sequence set forth in SEQ ID NO. 332; More preferably, the oligonucleotide comprises any one of a sense strand and an antisense strand combination selected from: (1) the sense strand comprises a sequence set forth in SEQ ID NO. 6, and the antisense strand comprises a sequence set forth in SEQ ID NO. 173; (2) the sense strand comprises a sequence set forth in SEQ ID NO. 20, and the antisense strand comprises a sequence set forth in SEQ ID NO. 187; (3) the sense strand comprises a sequence set forth in SEQ ID NO. 32, and the antisense strand comprises a sequence set forth in SEQ ID NO. 199; (4) the sense strand comprises a sequence set forth in SEQ ID NO. 38, and the antisense strand comprises a sequence set forth in SEQ ID NO. 205; (5) the sense strand comprises a sequence set forth in SEQ ID NO. 70, and the antisense strand comprises a sequence set forth in SEQ ID NO. 237; (6) the sense strand comprises a sequence set forth in SEQ ID NO. 96, and the antisense strand comprises a sequence set forth in SEQ ID NO. 263; (7) the sense strand comprises a sequence set forth in SEQ ID NO. 99, and the antisense strand comprises a sequence set forth in SEQ ID NO. 266; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 128, and the antisense strand comprises a sequence set forth in SEQ ID NO. 295; (9) the sense strand comprises a sequence set forth in SEQ ID NO. 130, and the antisense strand comprises a sequence set forth in SEQ ID NO. 297; (10) the sense strand comprises a sequence set forth in SEQ ID NO. 150, and the antisense strand comprises a sequence set forth in SEQ ID NO. 317; and (11) the sense strand comprises a sequence set forth in SEQ ID NO. 165, and the antisense strand comprises a sequence set forth in SEQ ID NO.

332.

7. The oligonucleotide or pharmaceutically acceptable salt thereof of any one of claims 1-6, wherein, the oligonucleotide comprises any one of the following sense and antisense strand combinations: (1) the sense strand comprises a sequence set forth in SEQ ID NO. 339, and the antisense strand comprises a sequence set forth in SEQ ID NO. 541; (2) the sense strand comprises a sequence set forth in SEQ ID NO. 340, and the antisense strand comprises a sequence set forth in SEQ ID NO. 542; (3) the sense strand comprises a sequence set forth in SEQ ID NO. 341, and the antisense strand comprises a sequence set forth in SEQ ID NO. 543; (4) the sense strand comprises a sequence set forth in SEQ ID NO. 343, and the antisense strand comprises a sequence set forth in SEQ ID NO. 545; (5) the sense strand comprises a sequence set forth in SEQ ID NO. 344, and the antisense strand comprises a sequence set forth in SEQ ID NO. 546; (6) the sense strand comprises a sequence set forth in SEQ ID NO. 345, and the antisense strand comprises a sequence set forth in SEQ ID NO. 547; (7) the sense strand comprises a sequence set forth in SEQ ID NO. 346, and the antisense strand comprises a sequence set forth in SEQ ID NO. 548; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 349, and the antisense strand comprises a sequence set forth in SEQ ID NO. 551; (9) the sense strand comprises a sequence set forth in SEQ ID NO. 351, and the antisense strand comprises a sequence set forth in SEQ ID NO. 553; (10) the sense strand comprises a sequence set forth in SEQ ID NO. 352, and the antisense strand comprises a sequence set forth in SEQ ID NO. 554; (11) the sense strand comprises a sequence set forth in SEQ ID NO. 353, and the antisense strand comprises a sequence set forth in SEQ ID NO. 555; (12) the sense strand comprises a sequence set forth in SEQ ID NO. 354, and the antisense strand comprises a sequence set forth in SEQ ID NO. 556; (13) the sense strand comprises a sequence set forth in SEQ ID NO. 355, and the antisense strand comprises a sequence set forth in SEQ ID NO. 557; (14) the sense strand comprises a sequence set forth in SEQ ID NO. 356, and the antisense strand comprises a sequence set forth in SEQ ID NO. 558; (15) the sense strand comprises a sequence set forth in SEQ ID NO. 357, and the antisense strand comprises a sequence set forth in SEQ ID NO. 559; (16) the sense strand comprises the sequence set forth in SEQ ID NO. 358, and the antisense strand comprises the sequence set forth in SEQ ID NO. 560; (17) the sense strand comprises the sequence set forth in SEQ ID NO. 360, and the antisense strand comprises the sequence set forth in SEQ ID NO. 562; (18) the sense strand comprises the sequence set forth in SEQ ID NO. 361, and the antisense strand comprises the sequence set forth in SEQ ID NO. 563; (19) the sense strand comprises the sequence set forth in SEQ ID NO. 366, and the antisense strand comprises the sequence set forth in SEQ ID NO. 568; (20) the sense strand comprises the sequence set forth in SEQ ID NO. 371, and the antisense strand comprises the sequence set forth in SEQ ID NO. 573; (21) the sense strand comprises the sequence set forth in SEQ ID NO. 372, and the antisense strand comprises the sequence set forth in SEQ ID NO. 574; (22) the sense strand comprises the sequence set forth in SEQ ID NO. 402, and the antisense strand comprises the sequence set forth in SEQ ID NO. 604; (23) the sense strand comprises the sequence set forth in SEQ ID NO. 403, and the antisense strand comprises the sequence set forth in SEQ ID NO. 605; (24) the sense strand comprises the sequence set forth in SEQ ID NO. 404, and the antisense strand comprises the sequence set forth in SEQ ID NO. 606; (25) the sense strand comprises the sequence set forth in SEQ ID NO. 405, and the antisense strand comprises the sequence set forth in SEQ ID NO. 607; (26) the sense strand comprises the sequence set forth in SEQ ID NO. 406, and the antisense strand comprises the sequence set forth in SEQ ID NO. 608; (27) the sense strand comprises the sequence set forth in SEQ ID NO. 410, and the antisense strand comprises the sequence set forth in SEQ ID NO. 612; (28) the sense strand comprises the sequence set forth in SEQ ID NO. 411, and the antisense strand comprises the sequence set forth in SEQ ID NO. 613; (29) the sense strand comprises the sequence set forth in SEQ ID NO. 412, and the antisense strand comprises the sequence set forth in SEQ ID NO. 614; (30) the sense strand comprises the sequence set forth in SEQ ID NO. 416, and the antisense strand comprises the sequence set forth in SEQ ID NO. 618; (31) the sense strand comprises the sequence set forth in SEQ ID NO. 420, and the antisense strand comprises the sequence set forth in SEQ ID NO. 622; (32) the sense strand comprises the sequence set forth in SEQ ID NO. 422, and the antisense strand comprises the sequence set forth in SEQ ID NO. 624; (33) the sense strand comprises the sequence set forth in SEQ ID NO. 424, and the antisense strand comprises the sequence set forth in SEQ ID NO. 626; (34) the sense strand comprises the sequence set forth in SEQ ID NO. 426, and the antisense strand comprises the sequence set forth in SEQ ID NO. 628; (35) the sense strand comprises the sequence set forth in SEQ ID NO. 427, and the antisense strand comprises the sequence set forth in SEQ ID NO. 629; (36) the sense strand comprises the sequence set forth in SEQ ID NO. 431, and the antisense strand comprises the sequence set forth in SEQ ID NO. 633; (37) the sense strand comprises the sequence set forth in SEQ ID NO. 434, and the antisense strand comprises the sequence set forth in SEQ ID NO. 636; (38) the sense strand comprises the sequence set forth in SEQ ID NO. 435, and the antisense strand comprises the sequence set forth in SEQ ID NO. 637; (39) the sense strand comprises the sequence set forth in SEQ ID NO. 437, and the antisense strand comprises the sequence set forth in SEQ ID NO. 639; (40) the sense strand comprises the sequence set forth in SEQ ID NO. 438, and the antisense strand comprises the sequence set forth in SEQ ID NO. 640; (41) the sense strand comprises the sequence set forth in SEQ ID NO. 442, and the antisense strand comprises the sequence set forth in SEQ ID NO. 644; (42) the sense strand comprises the sequence set forth in SEQ ID NO. 450, and the antisense strand comprises the sequence set forth in SEQ ID NO. 652; (43) the sense strand comprises the sequence set forth in SEQ ID NO. 453, and the antisense strand comprises the sequence set forth in SEQ ID NO. 655; (44) the sense strand comprises the sequence set forth in SEQ ID NO. 466, and the antisense strand comprises the sequence set forth in SEQ ID NO. 668; (45) the sense strand comprises the sequence set forth in SEQ ID NO. 482, and the antisense strand comprises the sequence set forth in SEQ ID NO. 684; (46) the sense strand comprises the sequence set forth in SEQ ID NO. 484, and the antisense strand comprises the sequence set forth in SEQ ID NO. 686; (47) the sense strand comprises the sequence set forth in SEQ ID NO. 494, and the antisense strand comprises the sequence set forth in SEQ ID NO. 696; (48) the sense strand comprises the sequence set forth in SEQ ID NO. 504, and the antisense strand comprises the sequence set forth in SEQ ID NO. 706; (49) the sense strand comprises the sequence set forth in SEQ ID NO. 505, and the antisense strand comprises the sequence set forth in SEQ ID NO. 707; (50) the sense strand comprises the sequence set forth in SEQ ID NO. 506, and the antisense strand comprises the sequence set forth in SEQ ID NO. 708; (51) the sense strand comprises the sequence set forth in SEQ ID NO. 508, and the antisense strand comprises the sequence set forth in SEQ ID NO. 710; (52) the sense strand comprises the sequence set forth in SEQ ID NO. 519, and the antisense strand comprises the sequence set forth in SEQ ID NO. 721; (53) the sense strand comprises the sequence set forth in SEQ ID NO. 522, and the antisense strand comprises the sequence set forth in SEQ ID NO. 724; (54) the sense strand comprises the sequence set forth in SEQ ID NO. 523, and the antisense strand comprises the sequence set forth in SEQ ID NO. 725; (55) the sense strand comprises the sequence set forth in SEQ ID NO. 524, and the antisense strand comprises the sequence set forth in SEQ ID NO. 726; (56) the sense strand comprises the sequence set forth in SEQ ID NO. 525, and the antisense strand comprises the sequence set forth in SEQ ID NO. 727; (57) the sense strand comprises the sequence set forth in SEQ ID NO. 526, and the antisense strand comprises the sequence set forth in SEQ ID NO. 728; (58) the sense strand comprises the sequence set forth in SEQ ID NO. 527, and the antisense strand comprises the sequence set forth in SEQ ID NO. 729; (59) the sense strand comprises the sequence set forth in SEQ ID NO. 530, and the antisense strand comprises the sequence set forth in SEQ ID NO. 732; (60) the sense strand comprises the sequence set forth in SEQ ID NO. 531, and the antisense strand comprises the sequence set forth in SEQ ID NO. 733; (61) the sense strand comprises the sequence set forth in SEQ ID NO. 532, and the antisense strand comprises the sequence set forth in SEQ ID NO. 734; (62) the sense strand comprises the sequence set forth in SEQ ID NO. 533, and the antisense strand comprises the sequence set forth in SEQ ID NO. 735; (63) the sense strand comprises the sequence set forth in SEQ ID NO. 535, and the antisense strand comprises the sequence set forth in SEQ ID NO. 737; (64) the sense strand comprises the sequence set forth in SEQ ID NO. 433, and the antisense strand comprises the sequence set forth in SEQ ID NO. 635; (65) the sense strand comprises the sequence set forth in SEQ ID NO. 739, and the antisense strand comprises the sequence set forth in SEQ ID NO. 757; (66) the sense strand comprises the sequence set forth in SEQ ID NO. 740, and the antisense strand comprises the sequence set forth in SEQ ID NO. 758; (67) the sense strand comprises a sequence as set forth in SEQ ID NO. 741, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 759; (68) the sense strand comprises a sequence as set forth in SEQ ID NO. 742, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 760; (69) the sense strand comprises a sequence as set forth in SEQ ID NO. 743, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 761 ; (70) the sense strand comprises a sequence as set forth in SEQ ID NO. 744, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 762; (71) the sense strand comprises a sequence as set forth in SEQ ID NO. 745, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 763; (72) the sense strand comprises a sequence as set forth in SEQ ID NO. 746, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 764; (73) the sense strand comprises a sequence as set forth in SEQ ID NO. 749, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 767; (74) the sense strand comprises a sequence as set forth in SEQ ID NO. 750, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 768; (75) the sense strand comprises a sequence as set forth in SEQ ID NO. 751, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 769; (76) the sense strand comprises a sequence as set forth in SEQ ID NO. 752, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 770; (77) the sense strand comprises a sequence as set forth in SEQ ID NO. 753, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 771 ; (78) the sense strand comprises a sequence as set forth in SEQ ID NO. 754, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 772; (79) the sense strand comprises a sequence as set forth in SEQ ID NO. 755, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 773; and (80) the sense strand comprises a sequence as set forth in SEQ ID NO. 756, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 774; Preferably, the oligonucleotide comprises any one of a sense strand and an antisense strand combination selected from: (1) the sense strand comprises a sequence as set forth in SEQ ID NO. 354, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 556; (2) the sense strand comprises a sequence as set forth in SEQ ID NO. 371, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 509; (4) the sense strand comprises a sequence as set forth in SEQ ID NO. 426, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 628; (3) the sense strand comprises the sequence set forth in SEQ ID NO. 427, and the antisense strand comprises the sequence set forth in SEQ ID NO. 629; (5) the sense strand comprises the sequence set forth in SEQ ID NO. 431, and the antisense strand comprises the sequence set forth in SEQ ID NO. 633; (6) the sense strand comprises the sequence set forth in SEQ ID NO. 434, and the antisense strand comprises the sequence set forth in SEQ ID NO. 636; (7) the sense strand comprises the sequence set forth in SEQ ID NO. 435, and the antisense strand comprises the sequence set forth in SEQ ID NO. 637; (8) the sense strand comprises the sequence set forth in SEQ ID NO. 437, and the antisense strand comprises the sequence set forth in SEQ ID NO. 639; (9) the sense strand comprises the sequence set forth in SEQ ID NO. 438, and the antisense strand comprises the sequence set forth in SEQ ID NO. 640; (10) the sense strand comprises the sequence set forth in SEQ ID NO. 442, and the antisense strand comprises the sequence set forth in SEQ ID NO. 644; (11) the sense strand comprises the sequence set forth in SEQ ID NO. 450, and the antisense strand comprises the sequence set forth in SEQ ID NO. 652; (12) the sense strand comprises the sequence set forth in SEQ ID NO. 453, and the antisense strand comprises the sequence set forth in SEQ ID NO. 655; (13) the sense strand comprises the sequence set forth in SEQ ID NO. 466, and the antisense strand comprises the sequence set forth in SEQ ID NO. 668; (14) the sense strand comprises the sequence set forth in SEQ ID NO. 482, and the antisense strand comprises the sequence set forth in SEQ ID NO. 684; (15) the sense strand comprises the sequence set forth in SEQ ID NO. 484, and the antisense strand comprises the sequence set forth in SEQ ID NO. 686; (16) the sense strand comprises the sequence set forth in SEQ ID NO. 494, and the antisense strand comprises the sequence set forth in SEQ ID NO. 696; (17) the sense strand comprises the sequence set forth in SEQ ID NO. 504, and the antisense strand comprises the sequence set forth in SEQ ID NO. 706; (18) the sense strand comprises the sequence set forth in SEQ ID NO. 505, and the antisense strand comprises the sequence set forth in SEQ ID NO. 707; (19) the sense strand comprises the sequence set forth in SEQ ID NO. 506, and the antisense strand comprises the sequence set forth in SEQ ID NO. 708; (20) the sense strand comprises the sequence set forth in SEQ ID NO. 508, and the antisense strand comprises the sequence set forth in SEQ ID NO. 710; (21) the sense strand comprises the sequence set forth in SEQ ID NO. 519, and the antisense strand comprises the sequence set forth in SEQ ID NO. 721; (22) the sense strand comprises the sequence set forth in SEQ ID NO. 522, and the antisense strand comprises the sequence set forth in SEQ ID NO. 724; (23) the sense strand comprises the sequence set forth in SEQ ID NO. 523, and the antisense strand comprises the sequence set forth in SEQ ID NO. 725; (24) the sense strand comprises the sequence set forth in SEQ ID NO. 526, and the antisense strand comprises the sequence set forth in SEQ ID NO. 728; (25) the sense strand comprises the sequence set forth in SEQ ID NO. 527, and the antisense strand comprises the sequence set forth in SEQ ID NO. 729; (26) the sense strand comprises the sequence set forth in SEQ ID NO. 532, and the antisense strand comprises the sequence set forth in SEQ ID NO. 734; (27) the sense strand comprises the sequence set forth in SEQ ID NO. 535, and the antisense strand comprises the sequence set forth in SEQ ID NO. 737; (28) the sense strand comprises the sequence set forth in SEQ ID NO. 433, and the antisense strand comprises the sequence set forth in SEQ ID NO. 635; (29) the sense strand comprises the sequence set forth in SEQ ID NO. 525, and the antisense strand comprises the sequence set forth in SEQ ID NO. 727; (30) the sense strand comprises the sequence set forth in SEQ ID NO. 533, and the antisense strand comprises the sequence set forth in SEQ ID NO. 735; (31) the sense strand comprises the sequence set forth in SEQ ID NO. 739, and the antisense strand comprises the sequence set forth in SEQ ID NO. 757; (32) the sense strand comprises the sequence set forth in SEQ ID NO. 740, and the antisense strand comprises the sequence set forth in SEQ ID NO. 758; (33) the sense strand comprises the sequence set forth in SEQ ID NO. 741, and the antisense strand comprises the sequence set forth in SEQ ID NO. 759; (34) the sense strand comprises the sequence set forth in SEQ ID NO. 742, and the antisense strand comprises the sequence set forth in SEQ ID NO. 760; (35) the sense strand comprises the sequence set forth in SEQ ID NO. 743, and the antisense strand comprises the sequence set forth in SEQ ID NO. 761; (36) the sense strand comprises the sequence set forth in SEQ ID NO. 744, and the antisense strand comprises the sequence set forth in SEQ ID NO. 762; (37) the sense strand comprises the sequence set forth in SEQ ID NO. 745, and the antisense strand comprises the sequence set forth in SEQ ID NO. 763; (38) the sense strand comprises a sequence set forth in SEQ ID NO. 746, and the antisense strand comprises a sequence set forth in SEQ ID NO. 764; (39) the sense strand comprises a sequence set forth in SEQ ID NO. 749, and the antisense strand comprises a sequence set forth in SEQ ID NO. 767; (40) the sense strand comprises a sequence set forth in SEQ ID NO. 750, and the antisense strand comprises a sequence set forth in SEQ ID NO. 768; (41) the sense strand comprises a sequence set forth in SEQ ID NO. 751, and the antisense strand comprises a sequence set forth in SEQ ID NO. 769; (42) the sense strand comprises a sequence set forth in SEQ ID NO. 752, and the antisense strand comprises a sequence set forth in SEQ ID NO. 770; (43) the sense strand comprises a sequence set forth in SEQ ID NO. 753, and the antisense strand comprises a sequence set forth in SEQ ID NO. 771; (44) the sense strand comprises a sequence set forth in SEQ ID NO. 754, and the antisense strand comprises a sequence set forth in SEQ ID NO. 772; (45) the sense strand comprises a sequence set forth in SEQ ID NO. 755, and the antisense strand comprises a sequence set forth in SEQ ID NO. 773; and (46) the sense strand comprises a sequence set forth in SEQ ID NO. 756, and the antisense strand comprises a sequence set forth in SEQ ID NO. 774; More preferably, the oligonucleotide comprises any one of a sense strand and an antisense strand combination selected from: (1) the sense strand comprises a sequence set forth in SEQ ID NO. 354, and the antisense strand comprises a sequence set forth in SEQ ID NO. 556; (2) the sense strand comprises a sequence set forth in SEQ ID NO. 427, and the antisense strand comprises a sequence set forth in SEQ ID NO. 629; (3) the sense strand comprises a sequence set forth in SEQ ID NO. 431, and the antisense strand comprises a sequence set forth in SEQ ID NO. 633; (4) the sense strand comprises a sequence set forth in SEQ ID NO. 434, and the antisense strand comprises a sequence set forth in SEQ ID NO. 636; (5) the sense strand comprises a sequence set forth in SEQ ID NO. 435, and the antisense strand comprises a sequence set forth in SEQ ID NO. 637; (6) the sense strand comprises a sequence set forth in SEQ ID NO. 438, and the antisense strand comprises a sequence set forth in SEQ ID NO. 640; (7) the sense strand comprises a sequence set forth in SEQ ID NO. 522, and the antisense strand comprises a sequence set forth in SEQ ID NO. 724; (8) the sense strand comprises a sequence set forth in SEQ ID NO. 523, and the antisense strand comprises a sequence set forth in SEQ ID NO. 725; (9) the sense strand comprises a sequence as set forth in SEQ ID NO. 526, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 728; (10) the sense strand comprises a sequence as set forth in SEQ ID NO. 527, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 729; (11) the sense strand comprises a sequence as set forth in SEQ ID NO. 532, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 734; (12) the sense strand comprises a sequence as set forth in SEQ ID NO. 535, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 737; (13) the sense strand comprises a sequence as set forth in SEQ ID NO. 433, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 635; (14) the sense strand comprises a sequence as set forth in SEQ ID NO. 442, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 644; (15) the sense strand comprises a sequence as set forth in SEQ ID NO. 525, and the antisense strand comprises a sequence as set forth in SEQ ID NO. 727; and (16) the sense strand comprises a sequence as set forth in SEQ ID NO. 533, and the antisense strand comprises a sequence as set forth in SEQ ID NO.

735.

8. A conjugate or a pharmaceutically acceptable salt thereof for use in inhibiting expression of INHBE, comprising: (i) an oligonucleotide of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the oligonucleotide or the pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands; Preferably, the targeting ligand comprises a carbohydrate, an aminosugar, a cholesterol, a polypeptide, or a lipid; Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; Preferably, the GalNac moiety is a monovalent GalNAc moiety, a bivalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety; Preferably, the targeting ligand is L96; 9. A composition comprising the oligonucleotide of any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or the conjugate of claim 8, and optionally a pharmaceutically acceptable carrier; Preferably, the dosage form of the composition is an oral dosage, an intravenous injection, a subcutaneous injection, or an intramuscular injection, preferably a subcutaneous injection; Preferably, the composition further comprises another drug for treating or / and preventing an INHBE-related disorder, condition, and / or pathology.

10. Use of the oligonucleotide of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, or the composition of claim 9, in the manufacture of a medicament for treating or / and preventing an INHBE-related disorder, condition, and / or pathology. The INHBE-related diseases, disorders, and / or conditions include, but are not limited to, obesity, type II diabetes, hyperlipidemia or dyslipidemia (elevated or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B, or other lipid moieties), obesity (particularly abdominal obesity), lipodystrophy (such as an inability to deposit fat locally in fat depots (partial lipodystrophy) or an inability to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenge, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis, or liver inflammation), nonalcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver damage, other markers of inflammation or fat deposition in the liver that are higher or elevated or altered, higher and / or high blood pressure, higher or high blood glucose or blood sugar, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the above conditions.

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