Specifically modified double-stranded oligonucleotide and composition thereof

Abstract

Provided are a specifically modified double-stranded oligonucleotide and a composition thereof. Further provided is a double-stranded oligonucleotide for inhibiting the expression of a target gene. The double-stranded oligonucleotide comprises a sense strand and an antisense strand complementary to at least a portion of mRNA of the target gene, wherein the length of the sense strand and the length of the antisense strand are each independently 15-30 nucleotides, the sense strand and the antisense strand are at least partially reversely complementary to form a double-stranded region, each nucleotide of the sense strand and the antisense strand is a modified nucleotide, and the double-stranded oligonucleotide comprises at least one modified internucleotide bond; the modified nucleotide comprises at least a 2'-methoxy-modified nucleotide or a 2'-fluoro-modified nucleotide. Compared with an industry-recognized modification model, the modified combination obtained by screening has lower toxicity, higher activity and stability, and a longer-lasting effect, and can reduce the off-target activity of siRNA to a certain extent. Moreover, experimental results show that the modified combination has high versatility.

Description

Specifically modified double-stranded oligonucleotides and compositions thereof TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and specifically relates to a specifically modified double-stranded oligonucleotide and a composition thereof. BACKGROUND

[0002] Exogenous synthetic small interfering RNA (siRNA) can inhibit gene expression through endogenous RNAi pathway to achieve post-transcriptional silencing. In 1998, Fire and Mello first revealed the phenomenon of RNA interference, which completely changed the understanding of human beings on the regulation of gene expression, and then proved that double-stranded RNA caused the phenomenon of RNA interference (Fire A, Nature, 1998; 391 (6669): 806-11.), and then the presence of siRNA was found in plants (Hamilton AJ, Science. 1999; 286 (5441): 950-2) and mammalian cells (Zamore PD, Tuschl T, Sharp PA. Cell. 2000; 101 (1): 25-33. Hammond SM. Nature. 2000; 404 (6775): 293-6.). By 2001, Elbashir et al. had successfully used synthetic siRNA for silencing, and determined the basic configuration and principle of siRNA and thus provided the basis for developing RNAi applications (Elbashir SM. Nature. 2001; 411 (6836): 494-8. Elbashir SM,. Nature. 2001; 411 (6836): 494-8.). Since then, hijacking endogenous selective silencing genes has become a widely used gene function research technology, and in addition, this method has shown impressive potential therapeutic effects.

[0003] Due to the fact that natural nucleotides are extremely susceptible to nuclease metabolic degradation in blood and tissues in vivo, and at the same time lack of suitable delivery systems, for a long time, the feasibility of siRNA as a drug has been greatly challenged, until the emergence of artificially modified ribonucleotides, which to some extent solve the problem of instability of siRNA in vivo, in addition, the emergence of LNP delivery technology and GalNAc delivery technology (see US8106022) makes the targeted delivery of siRNA a reality, and thus gives birth to the first LNP-siRNA drug Patisiran and GalNAc-siRNA drug Givosiran.

[0004] Commonly modified ribonucleotides are deoxyfluoro modification at position 2 of the furanose ring (2’-F) and methoxy modification (2’-oMe). The 2’-F modification has less impact on siRNA activity, but has potential toxicity. It has been reported that 2’-F modification of antisense oligonucleotides fully phosphorothioated resulted in acute hepatotoxicity in vivo by causing cellular protein reduction and double-stranded DNA breaks (Shen et al., Nucleic Acid Res., 43:4569-4578, 2015; Shen et al., NUCLEIC ACID RES., 46:2204-2217, 2018). To date, no evidence of such 2’-F modification has been observed in native RNAi oligonucleotides (Janas et al., NUCLEIC ACID THER., 26:363-371, 2016; Janas et al., NUCLEIC ACID THER., 27:11-22, 2016). Moreover, 2’-F siRNA is still well tolerated in clinical trials. Despite this, there is still a need to reduce the use of non-natural nucleoside analogs, such as 2’-F modified nucleosides, in siRNA. Unlike 2’-F, 2’-O-methyl RNA is a naturally occurring RNA modification found in tRNA and other small RNAs. 2’-oMe has stronger nuclease resistance due to greater steric hindrance, however, too much or inappropriate introduction of 2’-oMe in siRNA can interfere with siRNA loading with AGO2 protein and inhibit RNAi activity (Chiu et al., RNA, 9:1034-1048, 2003; Prakash et al., J. MED. CHEM., 48:4247-4253, 2005; Zheng et al., FASEB J., 27:4017-4026, 2013).

[0005] In summary, balancing the position and number of 2’-F and 2’-oMe on double-stranded siRNA to maximize its nuclease resistance without affecting its activity, while reducing toxic side effects, is an important direction for siRNA modification research and development. SUMMARY

[0006] To solve the problems in the prior art, the purpose of the present disclosure is to provide a specific modified double-stranded oligonucleotide and its composition, which maximizes its nuclease resistance without affecting its activity, while reducing toxic side effects.

[0007] In one aspect, the present disclosure provides a double-stranded oligonucleotide for inhibiting expression of a target gene, the double-stranded oligonucleotide comprising a sense strand and an antisense strand complementary to at least a portion of mRNA of the target gene, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region, each nucleotide of the sense strand and the antisense strand is a modified nucleotide, and the double-stranded oligonucleotide comprises at least one modified internucleotide linkage, the modified nucleotide comprises at least a 2'-methoxy modified nucleotide or a 2'-fluoro modified nucleotide,

[0008] the antisense strand of the double-stranded oligonucleotide comprises at least one of the following modifications:

[0009] (1) at least 4 2'-fluoro modified nucleotides;

[0010] (2) at least 8 2'-methoxy modified nucleotides;

[0011] (3) 2-4 modified internucleotide linkages.

[0012] and / or;

[0013] the sense strand of the double-stranded oligonucleotide comprises at least one of the following modifications:

[0014] (1) at least 1 2'-fluoro modified nucleotide;

[0015] (2) at least 11 2'-methoxy modified nucleotides;

[0016] (3) 2-4 modified internucleotide linkages.

[0017] In another aspect, the present disclosure provides a composition comprising the aforementioned double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0018] In another aspect, the present disclosure provides a cell comprising the aforementioned double-stranded oligonucleotide.

[0019] In another aspect, the present disclosure provides a method for inhibiting expression of a target gene in a cell, the method comprising the steps of:

[0020] delivering the aforementioned double-stranded oligonucleotide to the subject or the object to inhibit expression of a specific target gene in the subject.

[0021] In another aspect, the present disclosure provides a use of the aforementioned double-stranded oligonucleotide in the manufacture of a medicament for viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immune diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, pulmonary diseases, and rare diseases.

[0022] In another aspect, the present disclosure provides a method of treating or preventing a disease or disorder in a subject, wherein the method comprises administering to the subject the aforementioned oligonucleotide and / or the aforementioned composition in an amount sufficient to inhibit expression of a gene that causes the disease in the subject.

[0023] The present disclosure has at least the following beneficial effects:

[0024] The present disclosure, by arranging and combining 2'-F and 2'-oMe modifications of different nucleotides in the sense strand and the antisense strand of siRNA, preferably selects a modification combination type that has lower toxicity, higher activity and stability, and longer lasting effect than the modification mode generally recognized in the industry, and can reduce the off-target activity of siRNA to a certain extent. At the same time, the experimental results show that the modification combination type selected by the present disclosure has high universality. BRIEF DESCRIPTION OF DRAWINGS

[0025] FIGS. 1A-1F show IC50 of the cross combination of the sense strand and the antisense strand on TTR gene. 50 Detection results.

[0026] FIGS. 2A-2R show IC50 screening results of the cross combination of the sense strand and the antisense strand on human genes APP, AGT, and APOC3, respectively.

[0027] FIGS. 3A-3B show the verification results of the in vivo efficacy of different modification combinations.

[0028] FIGS. 4A-4E show the in vitro IC50 of the KM113P025G modification combination. 50 Data. DETAILED DESCRIPTION

[0029] I. DEFINITIONS

[0030] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the related terms are provided as follows.

[0031] As used herein, the term "about" or "approximately," as applied to one or more target values, means an amount that is similar to that recited. In certain embodiments, the term "approximately" or "about" means 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 in either direction (greater than or less than) of the recited reference value, unless otherwise stated or otherwise evident from context (unless such number would exceed 100% of a possible value). As used herein, "optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0032] 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.

[0033] As used herein, the term "siRNA" is a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary to it. siRNAs are typically double-stranded, comprising an antisense strand that is complementary to a target RNA, and a sense strand that is complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA (e.g., tRNA) and viral RNAs can also be targeted.

[0034] As used herein, the term "antisense strand" refers to one strand of an siRNA that comprises a region that is fully or substantially complementary to a target sequence.

[0035] 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, the term "region of complementarity" refers to a region on an antisense strand that is fully or substantially complementary to a target mRNA sequence. Where the region of complementarity is not fully complementary to the target sequence, mismatches can be located in the interior or in the terminal regions of the molecule. Generally, the most tolerated mismatches are located in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' and / or 3' end. The portion of the antisense strand that is most sensitive to mismatches is referred to as the "seed region." For example, in an siRNA comprising a 19 nt strand, the 19th position (from 5' to 3') can tolerate some mismatches. The terms "complementary," "fully complementary," and "substantially complementary" can be used with respect to base pairing between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0036] As used herein, the term "sense strand" refers to one strand of an siRNA that includes a region that is substantially complementary to a region that is an antisense strand as that term is defined herein.

[0037] As used herein, a "nucleoside" is a compound consisting of a purine or pyrimidine base, and either ribose or deoxyribose, a "nucleotide" is a compound consisting of a purine or pyrimidine base, ribose or deoxyribose, and a phosphate, and an "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) having, for example, a length of less than 100, 200, 300, or 400 nucleotides. An oligonucleotide can comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, 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.

[0038] As used herein, a "base" is a basic building block of synthetic nucleosides, nucleotides, and nucleic acids, which contains nitrogen in its constituent elements, also known as a "nitrogen-containing base." Herein, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of a nucleotide, which are adenine, uracil, thymine, guanine, and cytosine, respectively, with the specific structures as follows:

[0039] In some embodiments, m represents that the nucleotide adjacent to its left is a sugar ring 2'-OCH3 modified nucleotide, such as: Am, Um, Gm, and Cm represent A, U, G, and C with sugar ring 2'-OCH3 modification, respectively, with the specific structures as follows:

[0040] f represents that the nucleotide adjacent to its left is a sugar ring 2'-F modified nucleotide, such as: Af, Uf, Gf, and Cf represent A, U, G, and C with sugar ring 2'-f modification, respectively, with the specific structures as follows:

[0041] "s" represents that the two nucleotides adjacent to its left and right are linked by phosphorothioate, with the specific structure as follows:

[0042] wherein X represents: 2'-OCH3 or 2'-F modification.

[0043] L96 represents a GalNAc delivery carrier of the following structure well known in the art, wherein represents the position linked to siRNA by a phosphorothioate group or a phosphorothioate group, referring to PCT Publication Nos. WO2009073809 and WO2009082607.

[0044] In some embodiments, the GalNAc delivery carrier of the present disclosure is a GalNAc derivative, with the specific structure as follows:

[0045] 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) in addition to at the 2' position.

[0046] 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.

[0047] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that overhangs from the duplex structure of an siRNA. A nucleotide overhang is present, for example, when the 3 '-end of one strand of an siRNA extends beyond the 5 '-end of the other strand, or vice versa. An siRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, one or more nucleotides of an overhang can be present on the 5 '-end, the 3 '-end, or both ends of the antisense or sense strand of an siRNA.

[0048] "Blunt" or "blunt end" means that there are no unpaired nucleotides at the end of the double-stranded siRNA, i.e., no nucleotide overhang. A "blunt" siRNA is one that is double-stranded over its entire length, i.e., there is no nucleotide overhang at either end of the molecule.

[0049] Substantially all of the nucleotides of an iRNA of the application are modified. For example, substantially all of the nucleotides of the sense strand are modified nucleotides, and / or substantially all of the nucleotides of the antisense strand are modified nucleotides, and / or substantially all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. In other embodiments of the application, all of the nucleotides of an iRNA of the application are modified nucleotides. For example, all of the nucleotides of the sense strand are modified nucleotides, and / or all of the nucleotides of the antisense strand are modified nucleotides, and / or all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. Where "substantially all nucleotides are modified" means that the siRNA of the application is mostly but not entirely modified, and can include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0050] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657, while other carriers are known to those skilled in the art. Such formulations can typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can conveniently be used to prepare pharmaceutically acceptable salts, which are not excluded from the scope of the application. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts derived from hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like. In addition, pharmaceutically acceptable salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0051] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," and other similar terms, and includes inhibition at any level.

[0052] As used herein, the term "treatment" includes inhibiting, slowing, stopping, or reversing the progression or severity of an existing symptom or condition. Thus, treatment includes prevention, therapy, and / or cure. Furthermore, "therapeutic method," "treatment," "amelioration," or "prevention" can be used interchangeably herein. Prevention refers to preventing a potential disease and / or preventing the worsening of symptoms or development of the disease. As used herein, "therapeutic effect" means an effect resulting from the treatment of an individual that alters, usually improves, or ameliorates a symptom of a disease or disease condition, or cures the disease or disease condition. As used herein, "therapeutically effective amount" or "therapeutically effective dose" means an amount of a substance, compound, material, or composition comprising a compound that, after administration to a subject, is at least sufficient to produce a therapeutic effect. Thus, it is an amount necessary to prevent, cure, ameliorate, retard, or partially retard symptoms of a disease or disorder. As used herein, "prophylactically effective amount" or "prophylactically effective dose" means an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the intended prophylactic effect, e.g., prevent or delay the onset of a disease or symptoms, reduce the likelihood of the onset of a disease or symptoms. A fully prophylactically effective dose does not necessarily occur with the administration of one dose, and can only occur after the administration of a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0053] As used herein, the term "patient" refers to any animal, such as a mammal or a marsupial. The presently disclosed subject matter includes, but is not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any species of poultry.

[0054] As used herein, "prevent" and "prevention" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain "prophylactic benefit," a conjugate or composition can be administered to a patient at risk of suffering from a particular disease, or to a patient reporting one or more pathological symptoms of a disease, even though a diagnosis of this disease can not have been made.

[0055] II. DETAILED DESCRIPTION

[0056] In one aspect, the present disclosure provides a double-stranded oligonucleotide that inhibits expression of a target gene, the double-stranded oligonucleotide comprising a sense strand and an antisense strand that is complementary to at least a portion of an mRNA of the target gene, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region, each nucleotide of the sense strand and the antisense strand is a modified nucleotide, and the double-stranded oligonucleotide comprises at least one modified internucleotide linkage, the modified nucleotide comprises at least a 2'-methoxy modified nucleotide or a 2'-fluoro modified nucleotide,

[0057] The antisense strand of the double stranded oligonucleotide comprises at least one of the following modifications:

[0058] (1) at least 4 2’-fluoro modified nucleotides;

[0059] (2) at least 8 2’-methoxy modified nucleotides;

[0060] (3) 2-4 modified internucleotide linkages;

[0061] and / or;

[0062] The sense strand of the double stranded oligonucleotide comprises at least one of the following modifications:

[0063] (1) at least 1 2’-fluoro modified nucleotide;

[0064] (2) at least 11 2’-methoxy modified nucleotides;

[0065] (3) 2-4 modified internucleotide linkages.

[0066] In some embodiments, the sense strand and the antisense strand are substantially reverse complementary, substantially fully reverse complementary, or fully reverse complementary. Fully reverse complementary means that there are no mismatches between the two nucleotide sequences.

[0067] In some embodiments, the sense strand and the antisense strand are 15-25 nucleotides in length.

[0068] In some embodiments, the sense strand and the antisense strand are 17-21 nucleotides in length.

[0069] In some embodiments, the sense strand and the antisense strand are 19-21 nucleotides in length.

[0070] In some embodiments, the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length.

[0071] In some embodiments, the modified internucleotide linkage is a phosphorothioate linkage.

[0072] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises at least 4 2’-fluoro modified nucleotides, at least 8 2’-methoxy modified nucleotides.

[0073] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises at least 4 2’-fluoro modified nucleotides, at least 8 2’-methoxy modified nucleotides, and 4 phosphorothioate linkages.

[0074] In some embodiments, at least three of the nucleotides at positions 2, 6, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides. In some preferred embodiments, the nucleotides at positions 2, 6, 14, and 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides.

[0075] In some embodiments, at least seven of the nucleotides at positions 1, 9, 11, 13, 15, 17, 19, 21 from the 5' terminus of the antisense strand are 2'-methoxy-modified nucleotides.

[0076] In some preferred embodiments, the nucleotides at positions 1, 9, 11, 13, 15, 17, 19, and 21 from the 5' terminus of the antisense strand are 2'-methoxy-modified nucleotides.

[0077] In some embodiments, the antisense strand comprises at least two, three, four, or five consecutive 2'-fluoro-modified nucleotides at positions 3, 4, 5, 6, 7, 8 from the 5' terminus of the antisense strand.

[0078] In some embodiments, the phosphorothioate linkage is present between:

[0079] (1) the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 5' terminus of the antisense strand; and / or

[0080] (2) the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 3' terminus of the antisense strand.

[0081] In some embodiments, the phosphorothioate linkage is present between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 5' terminus of the antisense strand, and between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 3' terminus of the antisense strand.

[0082] In some embodiments, the phosphorothioate linkage is present between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the 19th and 20th nucleotides, the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0083] In some embodiments, the antisense strand of the double-stranded oligonucleotide comprises 6 2'-fluoro-modified nucleotides, 15 2'-methoxy-modified nucleotides.

[0084] In some embodiments, the antisense strand of the double-stranded oligonucleotide comprises 6 2'-fluoro-modified nucleotides, 15 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0085] In some embodiments, the nucleotides at positions 2, 5, 6, 7, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0086] In some embodiments, the nucleotides at positions 2, 5, 6, 7, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0087] In some embodiments, the antisense strand of the double-stranded oligonucleotide comprises 7 2'-fluoro-modified nucleotides, 14 2'-methoxy-modified nucleotides.

[0088] In some embodiments, the antisense strand of the double-stranded oligonucleotide comprises 7 2'-fluoro-modified nucleotides, 14 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0089] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0090] (1) the nucleotides at positions 2, 4, 6, 7, 14, 16, 20 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0091] (2) the nucleotides at positions 2, 5, 6, 7, 14, 16, 20 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0092] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0093] (1) the nucleotides at positions 2, 4, 6, 7, 14, 16, 20 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0094] (2) the 2nd, 5th, 6th, 7th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0095] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 8 2'-fluoro-modified nucleotides, 13 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0096] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 8 2'-fluoro-modified nucleotides, 13 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0097] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0098] (1) the 2nd, 3rd, 4th, 5th, 6th, 8th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0099] (2) the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0100] (3) the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0101] (4) the 2nd, 4th, 6th, 7th, 8th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0102] (5) the 2nd, 4th, 6th, 7th, 12th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0103] (6) the 2nd, 3rd, 6th, 7th, 12th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or

[0104] (7) the 2nd, 4th, 5th, 6th, 10th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0105] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0106] (1) the 2nd, 3rd, 4th, 5th, 6th, 8th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0107] (2) the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, and 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0108] (3) the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0109] (4) the 2nd, 4th, 6th, 7th, 8th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0110] (5) the 2nd, 4th, 6th, 7th, 12th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0111] (6) the 2nd, 3rd, 6th, 7th, 12th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0112] (7) the 2nd, 4th, 5th, 6th, 10th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0113] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 9 2'-fluoro-modified nucleotides, 12 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0114] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 9 2'-fluoro-modified nucleotides, 12 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0115] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0116] (1) the 2nd, 3rd, 5th, 6th, 8th, 10th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0117] (2) the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0118] (3) the nucleotides at positions 2, 4, 5, 6, 7, 12, 14, 16, and 20 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0119] (4) the nucleotides at positions 2, 3, 4, 5, 6, 10, 12, 14, and 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0120] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0121] (1) the nucleotides at positions 2, 3, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0122] (2) the nucleotides at positions 2, 3, 4, 5, 6, 7, 12, 14, 16, and 20 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0123] (3) the nucleotides at positions 2, 4, 5, 6, 7, 12, 14, 16, and 20 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0124] (4) the nucleotides at positions 2, 3, 4, 5, 6, 10, 12, 14, and 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0125] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 10 2'-fluoro-modified nucleotides, 11 2'-methoxy-modified nucleotides.

[0126] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 10 2'-fluoro-modified nucleotides, 11 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0127] In some embodiments, the nucleotides at positions 2, 3, 4, 5, 6, 8, 12, 14, 16, 18 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0128] In some embodiments, the nucleotides at positions 2, 3, 4, 5, 6, 8, 12, 14, 16, 18 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0129] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 11 2'-fluoro-modified nucleotides, 10 2'-methoxy-modified nucleotides.

[0130] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 11 2'-fluoro-modified nucleotides, 10 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0131] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0132] (1) the nucleotides at positions 2, 3, 5, 6, 7, 10, 12, 14, 16, 18, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0133] (2) the nucleotides at positions 2, 3, 5, 6, 7, 8, 10, 12, 14, 16, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0134] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0135] (1) the 2nd, 3rd, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0136] (2) the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0137] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 12 2'-fluoro-modified nucleotides, 9 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0138] In some embodiments, the antisense strand of the double stranded oligonucleotide comprises 12 2'-fluoro-modified nucleotides, 9 2'-methoxy-modified nucleotides, and 4 phosphorothioate linkages.

[0139] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0140] (1) the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, 18th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0141] (2) the 2nd, 3rd, 4th, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0142] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0143] (1) the nucleotides at positions 2, 3, 5, 6, 7, 8, 10, 12, 14, 16, 18, and 20 from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminus of the antisense strand; or

[0144] (2) the nucleotides at positions 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, and 20 from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminus of the antisense strand.

[0145] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0146] (1) the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, and 16 from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides;

[0147] (2) the nucleotides at positions 2, 5, 6, 7, 14, and 16 from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides;

[0148] (3) the nucleotides at positions 2, 4, 5, 6, 12, 14, 16, and 18 from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides; or

[0149] (4) the nucleotides at positions 2, 3, 4, 5, 6, 7, 12, 14, and 16 from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides.

[0150] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0151] (1) the 2nd, 4th, 6th, 7th, 10th, 12th, 14thand 16thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides and the remaining nucleotides are 2'-methoxy-modified nucleotides; and phosphorothioate linkages are present between the 1stand 2nd, 2ndand 3rd, 19thand 20th, and 20thand 21stnucleotides from the 5' terminus of the antisense strand;

[0152] (2) the 2nd, 5th, 6th, 7th, 14thand 16thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides and the remaining nucleotides are 2'-methoxy-modified nucleotides; and phosphorothioate linkages are present between the 1stand 2nd, 2ndand 3rd, 19thand 20th, and 20thand 21stnucleotides from the 5' terminus of the antisense strand;

[0153] (3) the 2nd, 4th, 5th, 6th, 12th, 14th, 16thand 18thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides and the remaining nucleotides are 2'-methoxy-modified nucleotides; and phosphorothioate linkages are present between the 1stand 2nd, 2ndand 3rd, 19thand 20th, and 20thand 21stnucleotides from the 5' terminus of the antisense strand; or

[0154] (4) the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14thand 16thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides and the remaining nucleotides are 2'-methoxy-modified nucleotides; and phosphorothioate linkages are present between the 1stand 2nd, 2ndand 3rd, 19thand 20th, and 20thand 21stnucleotides from the 5' terminus of the antisense strand.

[0155] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0156] (1) the 2nd, 4th, 6th, 7th, 10th, 12th, 14thand 16thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0157] (2) the 2nd, 5th, 6th, 7th, 14thand 16thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0158] In some embodiments, the antisense strand is selected from any one of the following combinations:

[0159] (1) the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; and phosphorothioate linkages exist between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0160] (2) the 2nd, 5th, 6th, 7th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; and phosphorothioate linkages exist between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0161] In some embodiments, the sense strand of the double-stranded oligonucleotide comprises at least 1 2'-fluoro-modified nucleotide, at least 11 2'-methoxy-modified nucleotides.

[0162] In some embodiments, the sense strand of the double-stranded oligonucleotide comprises at least 1 2'-fluoro-modified nucleotide, at least 11 2'-methoxy-modified nucleotides, and 2 or 4 phosphorothioate linkages.

[0163] In some embodiments, the nucleotide at the 9th position from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide.

[0164] In some embodiments, at least 10 of the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th positions from the 5' terminus of the sense strand are 2'-methoxy-modified nucleotides.

[0165] In some preferred embodiments, the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th positions from the 5' terminus of the sense strand are 2'-methoxy-modified nucleotides.

[0166] In some embodiments, at least 2 of the nucleotides at the 7th, 8th, and 9th positions from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and at least 1 of the nucleotides at the 5th, 6th, 10th, 11th, and 12th positions from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide.

[0167] In some embodiments, the phosphorothioate linkages exist between:

[0168] (1) between the 1st and 2nd, 2nd and 3rd nucleotides from the 5' terminus of the sense strand; and / or

[0169] (2) between the 1st and 2nd, 2nd and 3rd nucleotides from the 3' terminus of the sense strand.

[0170] In some preferred embodiments, the phosphorothioate linkage is between the 1st and 2nd, 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0171] In some embodiments, the sense strand of the double stranded oligonucleotide comprises 3 2'-fluoro modified nucleotides, 16 2'-methoxy modified nucleotides.

[0172] In some embodiments, the sense strand of the double stranded oligonucleotide comprises 3 2'-fluoro modified nucleotides, 16 2'-methoxy modified nucleotides and 2 phosphorothioate linkages.

[0173] In some embodiments, the nucleotides at positions 6, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro modified nucleotides and the remaining nucleotides are 2'-methoxy modified nucleotides.

[0174] In some embodiments, the nucleotides at positions 6, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro modified nucleotides and the remaining nucleotides are 2'-methoxy modified nucleotides and there is a phosphorothioate linkage between the 1st and 2nd, 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0175] In some embodiments, the sense strand of the double stranded oligonucleotide comprises 4 2'-fluoro modified nucleotides, 15 2'-methoxy modified nucleotides.

[0176] In some embodiments, the sense strand of the double stranded oligonucleotide comprises 4 2'-fluoro modified nucleotides, 15 2'-methoxy modified nucleotides and 2 phosphorothioate linkages.

[0177] In some embodiments, the sense strand is selected from any one of the following combinations:

[0178] (1) the nucleotides at positions 5, 6, 7, 9 from the 5' terminus of the sense strand are 2'-fluoro modified nucleotides and the remaining nucleotides are 2'-methoxy modified nucleotides;

[0179] (2) the nucleotides at positions 5, 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro modified nucleotides and the remaining nucleotides are 2'-methoxy modified nucleotides;

[0180] (3) the nucleotides at positions 5, 8, 9, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0181] (4) the nucleotides at positions 6, 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0182] (5) the nucleotides at positions 7, 8, 9, 10 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0183] (6) the nucleotides at positions 7, 8, 9, 11 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0184] (7) the nucleotides at positions 7, 8, 9, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0185] (8) the nucleotides at positions 8, 9, 10, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0186] In some embodiments, the sense strand is selected from any one of the following combinations:

[0187] (1) the nucleotides at positions 5, 6, 7, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand;

[0188] (2) the nucleotides at positions 5, 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand;

[0189] (3) the nucleotides at positions 5, 8, 9, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand;

[0190] (4) the 6th, 7th, 8th, and 9th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' end of the sense strand;

[0191] (5) the 7th, 8th, 9th, and 10th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' end of the sense strand;

[0192] (6) the 7th, 8th, 9th, and 11th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' end of the sense strand;

[0193] (7) the 7th, 8th, 9th, and 12th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; or

[0194] (8) the 8th, 9th, 10th, and 12th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

[0195] In some embodiments, the sense strand of the double-stranded oligonucleotide comprises 5 2'-fluoro-modified nucleotides and 14 2'-methoxy-modified nucleotides.

[0196] In some embodiments, the sense strand of the double-stranded oligonucleotide comprises 5 2'-fluoro-modified nucleotides, 14 2'-methoxy-modified nucleotides, and 2 phosphorothioate linkages.

[0197] In some embodiments, the 7th, 8th, 9th, 11th, and 12th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0198] In some embodiments, the nucleotides at positions 7, 8, 9, 11, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0199] In some embodiments, the sense strand of the double-stranded oligonucleotide comprises 6 2'-fluoro-modified nucleotides, and 13 2'-methoxy-modified nucleotides.

[0200] In some embodiments, the sense strand of the double-stranded oligonucleotide comprises 6 2'-fluoro-modified nucleotides, 13 2'-methoxy-modified nucleotides, and 2 phosphorothioate linkages.

[0201] In some embodiments, the sense strand is selected from any one of the following combinations:

[0202] (1) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0203] (2) the nucleotides at positions 6, 7, 8, 9, 11, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0204] In some embodiments, the sense strand is selected from any one of the following combinations:

[0205] (1) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; or

[0206] (2) the nucleotides at positions 6, 7, 8, 9, 11, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0207] In some embodiments, the sense strand of the double-stranded oligonucleotide comprises 7 2'-fluoro-modified nucleotides, and 12 2'-methoxy-modified nucleotides.

[0208] In some embodiments, the sense strand of the double stranded oligonucleotide comprises 7 2'-fluoro-modified nucleotides, 12 2'-methoxy-modified nucleotides, and 2 phosphorothioate linkages.

[0209] In some embodiments, the nucleotides at positions 5, 7, 8, 9, 10, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0210] In some embodiments, the nucleotides at positions 5, 7, 8, 9, 10, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

[0211] In some embodiments, the sense strand is selected from any one of the following combinations:

[0212] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0213] (2) the nucleotides at positions 7, 8, 9, 11 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0214] (3) the nucleotides at positions 7, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0215] (4) the nucleotides at positions 7, 8, 9, 10 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0216] (5) the nucleotides at positions 8, 9, 10, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0217] In some embodiments, the sense strand is selected from any one of the following combinations:

[0218] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand;

[0219] (2) the nucleotides at positions 7, 8, 9, 11 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand;

[0220] (3) the nucleotides at positions 7, 8, 9, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand;

[0221] (4) the nucleotides at positions 7, 8, 9, 10 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; or

[0222] (5) the nucleotides at positions 8, 9, 10, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0223] In some embodiments, the sense strand is selected from any one of the following combinations:

[0224] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0225] (2) the nucleotides at positions 7, 8, 9, 11 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0226] (3) the nucleotides at positions 7, 8, 9, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides.

[0227] In some embodiments, the sense strand is selected from any one of the following combinations:

[0228] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand;

[0229] (2) the nucleotides at positions 7, 8, 9, 11 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; or

[0230] (3) the nucleotides at positions 7, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

[0231] In some embodiments, the nucleotide at position 9 from the 5' end of the sense strand is a 2'-fluoro-modified nucleotide, and the remaining nucleotides are 2'-fluoro-modified or other modified nucleotides; the nucleotides at positions 2, 6, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-fluoro-modified or other modified nucleotides, and the other modified nucleotides include 2'-methoxy-modified nucleotides. Optionally, there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; and / or there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0232] In some embodiments, the nucleotide at the 9th position from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide, the remaining nucleotides are 2'-fluoro- or other modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the nucleotides at the 2nd, 6th, 14th, 16th positions from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, the remaining nucleotides are 2'-fluoro- or other modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, between the 20th and 21st nucleotides from the 5' terminus of the antisense strand, and the other modified nucleotides include 2'-methoxy-modified nucleotides.

[0233] In a preferred embodiment, the nucleotide at the 9th position from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are 2'-methoxy-modified nucleotides.

[0234] In a preferred embodiment, the nucleotide at the 9th position from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0235] In a preferred embodiment, the nucleotide at the 9th position from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are 2'-methoxy-modified nucleotides.

[0236] In a preferred embodiment, the nucleotide at the 9th position from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide, the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are 2'-methoxy-modified nucleotides, and phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0237] In a preferred embodiment, the nucleotide at position 9 from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at positions 1, 2, 3, 4, 13, 14, 15, 16, 17, 18, 19 are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 6, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 9, 11, 13, 15, 17, 19, 21 are 2'-methoxy-modified nucleotides. Alternatively, there is a phosphorothioate linkage between the first and second nucleotides, and between the second and third nucleotides from the 5' terminus of the sense strand; and / or there is a phosphorothioate linkage between the first and second nucleotides, between the second and third nucleotides, between the nineteenth and twentieth nucleotides, and between the twentieth and twenty-first nucleotides from the 5' terminus of the antisense strand.

[0238] In a preferred embodiment, the nucleotide at position 9 from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at positions 1, 2, 3, 4, 13, 14, 15, 16, 17, 18, 19 are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the first and second nucleotides, and between the second and third nucleotides from the 5' terminus of the sense strand; the nucleotides at positions 2, 6, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at positions 1, 9, 11, 13, 15, 17, 19, 21 are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the first and second nucleotides, between the second and third nucleotides, between the nineteenth and twentieth nucleotides, and between the twentieth and twenty-first nucleotides from the 5' terminus of the antisense strand.

[0239] In a preferred embodiment, at least two of the nucleotides at positions 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and at least one of the nucleotides at positions 5, 6, 10, 11, 12 is a 2'-fluoro-modified nucleotide; and at least two, three, four, or five consecutive nucleotides at positions 3, 4, 5, 6, 7, 8 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides. Alternatively, there is a phosphorothioate linkage between the first and second nucleotides, and between the second and third nucleotides from the 5' terminus of the sense strand; and / or there is a phosphorothioate linkage between the first and second nucleotides, between the second and third nucleotides, between the nineteenth and twentieth nucleotides, and between the twentieth and twenty-first nucleotides from the 5' terminus of the antisense strand.

[0240] In a preferred embodiment, at least two of the nucleotides at positions 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, at least one of the nucleotides at positions 5, 6, 10, 11, 12 is a 2'-fluoro-modified nucleotide, and there is a phosphorothioate linkage between the first and second nucleotides, and between the second and third nucleotides from the 5' end of the sense strand; at least two, three, four, or five consecutive 2'-fluoro-modified nucleotides at positions 3, 4, 5, 6, 7, 8 from the 5' end of the antisense strand, and there is a phosphorothioate linkage between the first and second nucleotides, between the second and third nucleotides, between the nineteenth and twentieth nucleotides, and between the twentieth and twenty-first nucleotides from the 5' end of the antisense strand.

[0241] In a preferred embodiment, the nucleotides at positions 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and at one and only one of positions 5, 11, or 12 is a 2'-fluoro-modified nucleotide; the nucleotides at positions 2, 6, 7, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and at one and only one of positions 4 or 5 is a 2'-fluoro-modified nucleotide. Optionally, there is a phosphorothioate linkage between the first and second nucleotides, and between the second and third nucleotides from the 5' end of the sense strand; and also optionally, there is a phosphorothioate linkage between the first and second nucleotides, between the second and third nucleotides, between the nineteenth and twentieth nucleotides, and between the twentieth and twenty-first nucleotides from the 5' end of the antisense strand.

[0242] In a preferred embodiment, the nucleotides at positions 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and at one and only one of positions 5, 11, or 12 is a 2'-fluoro-modified nucleotide, and there is a phosphorothioate linkage between the first and second nucleotides, and between the second and third nucleotides from the 5' end of the sense strand; the nucleotides at positions 2, 6, 7, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and at one and only one of positions 4 or 5 is a 2'-fluoro-modified nucleotide, and there is a phosphorothioate linkage between the first and second nucleotides, between the second and third nucleotides, between the nineteenth and twentieth nucleotides, and between the twentieth and twenty-first nucleotides from the 5' end of the antisense strand.

[0243] In some preferred embodiments, the sense strand is any one selected from the following combinations:

[0244] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0245] (2) the nucleotides at positions 7, 8, 9, 11 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0246] (3) the nucleotides at positions 7, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0247] and the antisense strand is any one selected from the following combinations:

[0248] (1) the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0249] (2) the nucleotides at positions 2, 5, 6, 7, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0250] Optionally, there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; and / or there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0251] In some embodiments, the double-stranded oligonucleotide has a 19-nucleotide sense strand and a 21-nucleotide antisense strand, and is any one selected from the following combinations:

[0252] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; and the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0253] (2) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0254] (3) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0255] (4) the 6th, 8th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 8th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0256] (5) the 7th, 8th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0257] (6) the 7th, 8th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 8th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0258] (7) the 5th, 6th, 7th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0259] (8) the 5th, 6th, 7th, 9th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0260] (9) the 5th, 7th, 8th, 9th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0261] (10) the 5th, 7th, 8th, 9th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 14th, 16th, 18th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0262] (11) the 5th, 7th, 8th, 9th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0263] (12) the 5th, 8th, 9th, 12th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0264] (13) the 6th, 7th, 8th, 9th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0265] (14) the 7th, 8th, 9th, 10th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0266] (15) the 7th, 8th, 9th, 11th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 14th, 16th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0267] (16) the 7th, 8th, 9th, 11th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0268] (17) the 7th, 8th, 9th, 11th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 7th, 12th, 14th, 16th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0269] (18) the 7th, 8th, 9th, 12th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 8th, 14th, 16th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0270] (19) the 7th, 8th, 9th, 12th nucleotides from the 5’ terminal of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 12th, 14th, 16th, 20th nucleotides from the 5’ terminal of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are all 2’-methoxy-modified nucleotides;

[0271] (20) the nucleotides at positions 7, 8, 9, 12 from the 5’ terminal end of the sense strand are -2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 5, 6, 7, 8, 10, 12, 14, 16, 20 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides;

[0272] (21) the nucleotides at positions 7, 8, 9, 12 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 5, 6, 7, 10, 12, 14, 16, 18, 20 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides;

[0273] (22) the nucleotides at positions 8, 9, 10, 12 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 4, 5, 6, 7, 12, 14, 16 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides;

[0274] (23) the nucleotides at positions 7, 8, 9, 11, 12 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 4, 5, 6, 10, 12, 14, 16 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides;

[0275] (24) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides;

[0276] (25) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 4, 5, 6, 7, 12, 14, 16 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides;

[0277] (26) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 5, 6, 8, 10, 12, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0278] (27) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0279] (28) the nucleotides at positions 6, 7, 8, 9, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 3, 6, 7, 12, 14, 16, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0280] (29) the nucleotides at positions 6, 7, 8, 9, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 5, 6, 10, 14, 16, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or

[0281] (30) the nucleotides at positions 5, 7, 8, 9, 10, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 5, 6, 12, 14, 16, 18 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides and the rest of the nucleotides are 2'-methoxy-modified nucleotides.

[0282] In some embodiments, in any one of the above groups (1)-(30), there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides, from the 5' end of the sense strand.

[0283] In some embodiments, in any one of groups (1)-(30) above, there are phosphorothioate linkages between the 1stand 2nd, 2ndand 3rd, 19thand 20th, 20thand 21stnucleotides from the 5' terminus of the antisense strand.

[0284] In some embodiments, the double-stranded oligonucleotide has a sense strand that is 19 nucleotides in length and an antisense strand that is 21 nucleotides in length, and the double-stranded oligonucleotide is any one selected from the following groups:

[0285] (1) the 5th, 7th, 8th, 9thnucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2nd, 2ndand 3rdnucleotides from the 5' terminus of the sense strand; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2nd, 2ndand 3rd, 19thand 20th, 20thand 21stnucleotides from the 5' terminus of the antisense strand;

[0286] (2) the 7th, 8th, 9th, 12thnucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2nd, 2ndand 3rdnucleotides from the 5' terminus of the sense strand; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16thnucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2nd, 2ndand 3rd, 19thand 20th, 20thand 21stnucleotides from the 5' terminus of the antisense strand;

[0287] (3) the 7th, 8th, 9th, 11st nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate bonds between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate bonds between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0288] (4) the 6th, 8th, 9th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate bonds between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the 2nd, 3rd, 4th, 5th, 6th, 8th, 14th, 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate bonds between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand;

[0289] (5) the 7th, 8th, 9th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate bonds between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate bonds between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand;

[0290] (6) the 7th, 8th, and 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 3rd, 5th, 6th, 8th, 10th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0291] (7) the 5th, 6th, 7th, and 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, and 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0292] (8) the 5th, 6th, 7th, and 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0293] (9) the nucleotides at the positions 5, 7, 8, 9 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ terminal end of the sense strand; the nucleotides at the positions 2, 3, 4, 5, 6, 7, 12, 14, 16 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminal end of the antisense strand;

[0294] (10) the nucleotides at the positions 5, 7, 8, 9 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ terminal end of the sense strand; the nucleotides at the positions 2, 3, 4, 5, 6, 8, 12, 14, 16, 18 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminal end of the antisense strand;

[0295] (11) the nucleotides at the positions 5, 7, 8, 9 from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ terminal end of the sense strand; the nucleotides at the positions 2, 3, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20 from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminal end of the antisense strand;

[0296] (12) the nucleotides at the positions 5, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the nucleotides at the positions 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand;

[0297] (13) the nucleotides at the positions 6, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the nucleotides at the positions 2, 3, 5, 6, 7, 10, 12, 14, 16, 18, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand;

[0298] (14) the nucleotides at the positions 7, 8, 9, 10 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the nucleotides at the positions 2, 4, 5, 6, 12, 14, 16, 18 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand;

[0299] (15) the 7th, 8th, 9th, and 11stnucleotides from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2ndnucleotides, and between the 2ndand 3rdnucleotides from the 5’ terminal end of the sense strand; the 2nd, 4th, 6th, 7th, 14th, 16th, and 20thnucleotides from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2ndnucleotides, between the 2ndand 3rdnucleotides, between the 19thand 20thnucleotides, and between the 20thand 21stnucleotides from the 5’ terminal end of the antisense strand;

[0300] (16) the 7th, 8th, 9th, and 11stnucleotides from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2ndnucleotides, and between the 2ndand 3rdnucleotides from the 5’ terminal end of the sense strand; the 2nd, 4th, 6th, 7th, 14th, 16th, and 20thnucleotides from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2ndnucleotides, between the 2ndand 3rdnucleotides, between the 19thand 20thnucleotides, and between the 20thand 21stnucleotides from the 5’ terminal end of the antisense strand;

[0301] (16) the 7th, 8th, 9th, and 11stnucleotides from the 5’ terminal end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2ndnucleotides, and between the 2ndand 3rdnucleotides from the 5’ terminal end of the sense strand; the 2nd, 4th, 6th, 7th, 14th, 16th, and 20thnucleotides from the 5’ terminal end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1stand 2ndnucleotides, between the 2ndand 3rdnucleotides, between the 19thand 20thnucleotides, and between the 20thand 21stnucleotides from the 5’ terminal end of the antisense strand;

[0302] (18) the 7th, 8th, 9th, and 12th nucleotides from the 5’ terminus of the sense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ terminus of the sense strand; the 2nd, 4th, 6th, 7th, 8th, 14th, 16th, and 20th nucleotides from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminus of the antisense strand;

[0303] (19) the 7th, 8th, 9th, and 12th nucleotides from the 5’ terminus of the sense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ terminus of the sense strand; the 2nd, 4th, 6th, 7th, 12th, 14th, 16th, and 20th nucleotides from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminus of the antisense strand;

[0304] (20) the 7th, 8th, 9th, and 12th nucleotides from the 5’ terminus of the sense strand are -2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ terminus of the sense strand; the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, and 20th nucleotides from the 5’ terminus of the antisense strand are 2’-fluoro-modified nucleotides, and the remaining nucleotides are 2’-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ terminus of the antisense strand;

[0305] (21) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 3rd, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0306] (22) the 8th, 9th, 10th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0307] (23) the 7th, 8th, 9th, 11th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 3rd, 4th, 5th, 6th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0308] (24) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5’ end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ end of the sense strand; the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5’ end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ end of the antisense strand;

[0309] (25) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5’ end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ end of the sense strand; the nucleotides at positions 2, 3, 4, 5, 6, 7, 12, 14, 16 from the 5’ end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ end of the antisense strand;

[0310] (26) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5’ end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ end of the sense strand; the nucleotides at positions 2, 3, 5, 6, 8, 10, 12, 14, 16 from the 5’ end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ end of the antisense strand;

[0311] (27) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5’ end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ end of the sense strand; the nucleotides at positions 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20 from the 5’ end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ end of the antisense strand;

[0312] (28) the nucleotides at positions 6, 7, 8, 9, 11, 12 from the 5’ end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ end of the sense strand; the nucleotides at positions 2, 3, 6, 7, 12, 14, 16, 20 from the 5’ end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ end of the antisense strand;

[0313] (29) the nucleotides at positions 6, 7, 8, 9, 11, 12 from the 5’ end of the sense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5’ end of the sense strand; the nucleotides at positions 2, 4, 5, 6, 10, 14, 16, 20 from the 5’ end of the antisense strand are 2’-fluoro-modified nucleotides, and the rest of the nucleotides are 2’-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5’ end of the antisense strand; or

[0314] (30) the nucleotides at positions 5, 7, 8, 9, 10, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the nucleotides at positions 2, 4, 5, 6, 12, 14, 16, 18 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0315] In preferred embodiments, the double-stranded oligonucleotide is selected from any one of the following combinations:

[0316] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0317] (2) the nucleotides at positions 7, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0318] (3) the nucleotides at positions 7, 8, 9, 11 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 5, 6, 7, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0319] (4) the nucleotides at positions 7, 8, 9, 10 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 5, 6, 12, 14, 16, 18 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0320] (5) the 8th, 9th, 10th, 12th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides.

[0321] In some embodiments, in any of (1)-(5) above, there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

[0322] In some embodiments, in any of (1)-(5) above, there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0323] In preferred embodiments, the double-stranded oligonucleotide is any one selected from the following combinations:

[0324] (1) the 5th, 7th, 8th, 9th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, between the 20th and 21st nucleotides from the 5' end of the antisense strand;

[0325] (2) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0326] (3) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand;

[0327] (4) the 7th, 8th, 9th, 10th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0328] (5) the 8th, 9th, 10th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0329] In more preferred embodiments, the double-stranded oligonucleotide is any one selected from the following combinations:

[0330] (1) the 5th, 7th, 8th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides;

[0331] (2) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or

[0332] (3) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides.

[0333] In some embodiments, in any one of the above (1)-(3), there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

[0334] In some embodiments, in any of groups (1)-(3) above, there are phosphorothioate linkages between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0335] In a preferred embodiment, the double-stranded oligonucleotide is any one selected from the following combinations:

[0336] (1) the 5th, 7th, 8th, 9th, 12th, 14th, 16th, and 18th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0337] (2) the 7th, 8th, 9th, 12th, 14th, 16th, and 18th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd, 2nd and 3rd, 19th and 20th, and 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0338] (3) the 7th, 8th, 9th, 11th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there are phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0339] In some embodiments, the modified nucleotide further comprises a nucleotide selected from the group consisting of: 2'-deoxynucleotide, 2',3'-seco nucleotide mimic, locked nucleotide acid (LNA), unlocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), bicyclic nucleic acid (BNA), 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribothymidine, inverted nucleotide, inverted abasic nucleotide (Invab), inverted 2'-OMe nucleotide, inverted 2'-deoxynucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, 3'-OMe nucleotide, phosphate group modified nucleotide, terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, 2'-amino-modified nucleotide, phosphoramidate, non-natural base comprising nucleotide, or 5'-phosphate mimic modified nucleotide.

[0340] In some embodiments, the 5'-phosphate mimic is selected from 5'-oxymethylphosphonate, 5'-vinylphosphonate, 5' vinylphosphonate-2'-N-acetyl, or 5'-malonylphosphonate.

[0341] In some embodiments, at least one nucleotide of the double-stranded oligonucleotide is conjugated to one or more targeting ligands.

[0342] In some embodiments, the targeting ligand is conjugated to the sense strand of the oligonucleotide.

[0343] In some embodiments, the targeting ligand is conjugated to any one nucleotide of the sense strand of the oligonucleotide.

[0344] In some embodiments, the targeting ligand is conjugated to the 5' end of the sense strand of the oligonucleotide.

[0345] In some embodiments, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3 '-end.

[0346] In some embodiments, the protein is selected from a natural protein, a synthetic polyamino acid, and an antibody.

[0347] In some embodiments, the natural protein is selected from human serum albumin, low density lipoprotein, or globulin.

[0348] In some embodiments, the synthetic polyamino acid is selected from polylysine, poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactic-co- ethylene glycol) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer, polyethylene glycol, polyvinyl alcohol, polyurethane, poly(2- ethylacrylic acid), N-isopropyl acrylamide polymer, or polyphosphazene.

[0349] In some embodiments, the carbohydrate is selected from dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, an aminosugar, or hyaluronic acid.

[0350] In some embodiments, the lipid is selected from a fatty acid, a sterol, or a phospholipid.

[0351] In some embodiments, the fatty acid is selected from decanoic acid, octanoic acid, lauric acid, palmitic acid, myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, or eicosapentaenoic acid.

[0352] In some embodiments, the sterol is selected from cholesterol, cholesteryl, cholestanol, stigmasterol, cholestanic acid, or ergosterol.

[0353] In some embodiments, the phospholipid is selected from di-hexadecyl-rac-glycerol or triethyl-amine 1,2-di-O-hexadecyl-rac-glycerol-3-hydrogen phosphonate.

[0354] In some embodiments, the targeting ligand comprises an aminosugar.

[0355] In some embodiments, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0356] In some embodiments, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0357] In some embodiments, the targeting ligand has the following structure: In some embodiments, the targeting ligand has the following structure:

[0358] In some embodiments, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3 '-end via a linker, forming a conjugate as follows:

[0359] X can be selected from S or O.

[0360] In some embodiments, the targeting ligand is Compound 2, which has the following structure:

[0361] 、

[0362] In some embodiments, the Compound 2 is conjugated to the sense strand of the oligonucleotide at the 3 '-end.

[0363] In some embodiments, the double-stranded oligonucleotide inhibits the expression of a target gene in a cell, the target gene being selected from the group consisting of: LPA, PNPLA3, ASGR1, F7, F12, FXI, APOCIII, APOB, APOL1, TTR, PCSK9, SCAP, KRAS, CD274, PDCD1, C5, C3, CFB, MASP2, ALAS1, MSTN, HAO1, LDHA, ANGPTL3, SERPINA1, HSD17B13, AGT, HAMP, LECT2, EGFR, VEGF, KIF11, AT3, CTNNB1, HMGB1, HIF1A, APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, SCN9A, SCN10A, CACNA1B, ATXNl, SCA1, SCA7, SCA8, MeCP2, PRNP, SODl, INHBE, CIDEB, DMPK, and STAT3.

[0364] In another aspect, the present disclosure provides a composition comprising the double-stranded oligonucleotide or a pharmaceutically acceptable salt thereof of the foregoing, and optionally a pharmaceutically acceptable carrier.

[0365] In some embodiments, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, 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, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and

[0366] These formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of the composition, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent of the composition. In certain embodiments, the formulations of the present application comprise an excipient selected from the group consisting of cyclodextrin, cellulose, liposome, micelle-forming agent (e.g., bile acid) and polymeric carrier (e.g., polyester and polyanhydride); and a compound of the present application. In certain embodiments, the foregoing formulations render the compounds of the present application orally bioavailable.

[0367] In some embodiments, the composition further comprises one or more additional therapeutic components.

[0368] In some embodiments, the composition is packaged in a kit, container, wrapper, dispenser, pre-filled syringe, or vial.

[0369] The compositions of the disclosure can be administered in a variety of ways, depending upon whether local or systemic treatment is desired, and upon the area to be treated. In some embodiments, the compositions are formulated for ocular, vaginal, rectal, intranasal, transdermal, subcutaneous administration, intravenous drip, intra-arterial, intralymphatic, intrabronchial, intrapleural, intraperitoneal, intracerebrospinal, or intramuscular injection, pulmonary, intrathecal, or intraventricular administration.

[0370] In some embodiments, the unit dose administered is less than 10 mg / kg of body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg / kg of body weight, and less than 200 nanomoles of RNA agent (e.g., about 4.4 x 1016copies) / kg of body weight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nanomoles of RNA agent / kg of body weight.

[0371] The determined amount can be an amount effective to treat or prevent a disease or disorder (e.g., a disease or disorder associated with a target RNA). The unit dose can be administered, for example, by injection (e.g., intravenously, subcutaneously, or intramuscularly), inhalation dose, or topical application. In some embodiments, the dose can be less than 10, 5, 2, 1, or 0.1 mg / kg of body weight.

[0372] In some embodiments, the unit dose is administered less frequently than once per day, e.g., less frequently than once every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a frequency (e.g., not at a regular frequency). For example, the unit dose can be administered a single time.

[0373] In another aspect, the disclosure provides a cell comprising the double-stranded oligonucleotide of any of the preceding.

[0374] In another aspect, the disclosure provides a method of inhibiting expression of a target gene in a cell, the method comprising the step of:

[0375] delivering the double-stranded oligonucleotide of the preceding or the composition of the preceding to the subject or subject to inhibit expression of a particular target gene in the subject.

[0376] In some embodiments, the target gene is selected from the group consisting of LPA, PNPLA3, ASGR1, F7, F12, FXI, APOCIII, APOB, APOL1, TTR, PCSK9, SCAP, KRAS, CD274, PDCD1, C5, C3, CFB, MASP2, ALAS1, MSTN, HAO1, LDHA, ANGPTL3, SERPINA1, HSD17B13, AGT, HAMP, LECT2, EGFR, VEGF, KIF11, AT3, CTNNB1, HMGB1, HIF1A, APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, SCN9A, SCN10A, CACNA1B, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, INHBE, CIDEB, DMPK, and STAT3.

[0377] In another aspect, the present disclosure provides use of the aforementioned double-stranded oligonucleotide and / or the aforementioned composition in the manufacture of a medicament for viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immunological diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, pulmonary diseases, tumors, and rare diseases.

[0378] In another aspect, the present disclosure provides a method of treating or preventing a disease or disorder in a subject, wherein the method comprises administering to the subject the aforementioned oligonucleotide and / or the aforementioned composition in an amount sufficient to inhibit expression of a gene that causes the disease in the subject. In some embodiments, the disease or disorder is selected from viral diseases, neuromuscular diseases, bacterial infections, inflammatory and immunological diseases, metabolic diseases, liver diseases, kidney diseases, cardiovascular diseases, ophthalmic diseases, pulmonary diseases, tumors, and rare diseases.

[0379] In another aspect, the present disclosure also provides a double-stranded oligonucleotide for inhibiting expression of Mannan-binding lectin-associated serine protease 2 (MASP2) in a cell, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region, the antisense strand is at least partially complementary to a segment of nucleotides in a MASP2 transcribed mRNA, the sense strand has a length of no more than 21 nucleotides, and the antisense strand has a length of no more than 23 nucleotides. In some embodiments, the sense strand has a length of 19 nucleotides, and the antisense strand has a length of 21 nucleotides.

[0380] In some embodiments, the double-stranded oligonucleotide comprises modified nucleotides.

[0381] In some embodiments, the nucleotides at positions 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and one and only one of the nucleotides at positions 5, 11, or 12 is a 2'-fluoro-modified nucleotide; preferably, the nucleotides at positions 2, 6, 7, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and one and only one of the nucleotides at positions 4 or 5 is a 2'-fluoro-modified nucleotide. Optionally, there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; also optionally, there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0382] In some embodiments, the nucleotides at positions 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and one and only one of the nucleotides at positions 5, 11, or 12 is a 2'-fluoro-modified nucleotide, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; preferably, the nucleotides at positions 2, 6, 7, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and one and only one of the nucleotides at positions 4 or 5 is a 2'-fluoro-modified nucleotide, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0383] In some embodiments, the modification type of the double-stranded oligonucleotide is selected from any of the following combinations:

[0384] (1) the nucleotides at positions 5, 7, 8, 9 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides;

[0385] (2) the nucleotides at positions 7, 8, 9, 12 from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or

[0386] (3) the 7th, 8th, 9th, 11th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides.

[0387] In some embodiments, in any one of the above groups (1)-(3), there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

[0388] In some embodiments, in any one of the above groups (1)-(3), there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, between the 20th and 21st nucleotides from the 5' end of the antisense strand.

[0389] In some embodiments, the modification type of the double-stranded oligonucleotide is selected from any one of the following combinations:

[0390] (1) the 5th, 7th, 8th, 9th nucleotides from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' end of the sense strand; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate linkage between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, between the 20th and 21st nucleotides from the 5' end of the antisense strand;

[0391] (2) the 7th, 8th, 9th, 12th nucleotide from the 5' terminus of the sense strand is 2'-fluoro-modified nucleotide, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate bond between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotide from the 5' terminus of the antisense strand is 2'-fluoro-modified nucleotide, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate bond between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand; or

[0392] (3) the 7th, 8th, 9th, 11th nucleotide from the 5' terminus of the sense strand is 2'-fluoro-modified nucleotide, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate bond between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotide from the 5' terminus of the antisense strand is 2'-fluoro-modified nucleotide, and the rest of the nucleotides are 2'-methoxy-modified nucleotides, and there is a phosphorothioate bond between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

[0393] In some embodiments, the double-stranded oligonucleotide that inhibits the expression of MASP2 can be, for example, an siRNA of the following:

[0394] the sense strand is the sequence set forth in SEQ ID NO: 267, and the antisense strand is the sequence set forth in SEQ ID NO: 264; or

[0395] the sense strand is the sequence set forth in SEQ ID NO: 262, and the antisense strand is the sequence set forth in SEQ ID NO: 264; or

[0396] the sense strand is the sequence set forth in SEQ ID NO: 266, and the antisense strand is the sequence set forth in SEQ ID NO: 264.

[0397] In some embodiments, wherein at least one nucleotide of the double-stranded oligonucleotide is conjugated to one or more targeting ligands; in some embodiments, the targeting ligand is conjugated to the sense strand of the oligonucleotide; preferably, the targeting ligand is conjugated to any one nucleotide of the sense strand of the oligonucleotide. In some embodiments, the targeting ligand is conjugated to the 5’ end of the sense strand of the oligonucleotide; or, the targeting ligand is conjugated to the 3’-end of the sense strand of the oligonucleotide.

[0398] In some preferred embodiments, 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.

[0399] In some embodiments, the targeting ligand has the following structure:

[0400] Preferably, the targeting ligand is conjugated to the 3’-end of the sense strand of the oligonucleotide.

[0401] Preferably, the targeting ligand is compound 2, which has the following structure:

[0402] Preferably, the compound 2 is conjugated to the 3’-end of the sense strand of the oligonucleotide.

[0403] In some aspects, the present disclosure also provides a pharmaceutical composition, a cell, a kit comprising the double-stranded oligonucleotide inhibiting the expression of MASP2 as described above.

[0404] In some aspects, the present disclosure also provides a pharmaceutical composition, a cell, a kit comprising the double-stranded oligonucleotide inhibiting the expression of MASP2 as described above.

[0405] In yet other aspects, the present disclosure also provides a method of inhibiting MASP2 gene expression in vitro, the method comprising contacting an effective amount of the double-stranded oligonucleotide as described above and / or the composition as described above with a cell.

[0406] For the purpose of clarity and brevity, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the present disclosure can include embodiments having combinations of all or some of the described features.

[0407] 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.

[0408] Examples

[0409] Example 1: Preparation of siRNA and conjugates thereof

[0410] Abbreviations for nucleotide monomers used in the nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are connected to one another by 5'-3' phosphodiester linkages, unless otherwise indicated. Preparation of L96 was performed according to the method described in patent CN104717982B.

[0411] The preparation process of the oligonucleotide is specifically as follows:

[0412] (1) Preparation of siRNA

[0413] The siRNA sequence was synthesized by LK-48 synthesizer (LinkBio) on a 500 nmol scale solid support by phosphoramidite chemistry. The solid support was controlled pore glass Auxiliary synthesis reagents and 2-cyanoethyl phosphoramidite monomers (2'-deoxy-2'-fluoro, 2'-O-methyl, RNA, DNA) were from Wuhu Huaren Technology Co., Ltd. or Beijing Dinaxingke Biotechnology Co., Ltd. Additional phosphoramidite monomers were obtained by commercial purchase, self-preparation or custom synthesis. The phosphoramidite monomers were provided in 50 mM acetonitrile or 1:1 acetonitrile: dichloromethane solution, the deprotection reagent was 3% trichloroacetic acid in dichloromethane solution, and the reaction time was 30 s with 4 deprotections. The activated agent used in the coupling reaction was 0.25 M 5-benzylthio tetrazole (BTT) in acetonitrile, and the reaction time was 160 s with 3 couplings. The 50 mM 1:1 acetonitrile: pyridine solution prepared using N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazole-5-yl) formamidine was used to generate the phosphorothioate linkage, and the oxidation time was 120 s with 2 oxidations. The capping agent used was 10% acetic anhydride in tetrahydrofuran and N-methylimidazole / pyridine / tetrahydrofuran solution, and the capping time was 20 s with 2 cappings. All sequences were deprotected with DMT off at the end.

[0414] After the completion of solid-phase synthesis, the solid-supported oligonucleotide was treated with 500 μL of 3:1 ammonia water: ethanol at 45 °C for 16 hours to cleave it from the solid support, followed by removal of additional labile protecting groups.

[0415] The oligonucleotide was precipitated with 10% v / v 5M NaCl solution and 3 volumes of absolute ethanol, centrifuged at 4 °C, and the supernatant was removed. The oligonucleotide was re-dissolved in dd-H2O and purified using an Agilent 1260 HPLC. The prepared solution was freeze-dried and desalted by salt exchange. Then, quantitative analysis was performed using LC-MS and UV spectroscopy. The OD 260 The concentration of the oligonucleotide solution was determined.

[0416] The sense strand and the antisense strand were mixed in an equimolar ratio, incubated in a water bath at 95 °C for 5 min, and then slowly returned to room temperature over 2-3 hours. The concentration and information of each double strand were confirmed for subsequent in vitro screening tests.

[0417] (2) Preparation of ligand-conjugated siRNA

[0418] The synthesis process of the ligand L96 (GalNAc) is referred to in PCT patents WO2009073809 and WO2009082607. The structural formula of the ligand L96 is as follows:

[0419] Further, the ligand is conjugated to the 3'-end of the sense strand of the oligonucleotide through a linker to form the following conjugate:

[0420] X can be selected from S or O.

[0421] Compound L96 was dissolved in DMF with TBTU (2 eq) followed by DIPA (4 eq) and shaken until the solution was clear. Blank support (1 eq, 160 pmol / g, 1.6 mg) was added and the reaction was shaken at 25 °C for 16 h. A 2 mg sample was taken and the DMT group was removed with 80 mL of 3% trichloroacetic acid / dichloromethane solution. The solution was passed through a spectrophotometer to determine the Abs and the loading was calculated to be 40-50 pmol / g. The reaction was filtered and the solid was dried under vacuum for 16 h. The solid was then used for solid phase synthesis.

[0422] After the solid support with custom GalNAc (L96) was weighed, it was packed into the empty column with the lower frit and the upper frit was placed on top. Ancillary synthesis reagents and 2-cyanoethyl phosphoramidite monomers (2’-deoxy-2’-fluoro, 2’-O-methyl, RNA, DNA) were obtained from Wuhu Huaren Technology Co., Ltd. or Beijing Dinaxinkexue Technology Co., Ltd. Additional phosphoramidite monomers were obtained by commercial purchase, prepared in-house, or custom synthesized. Phosphoramidite monomers were provided at 50 mM in acetonitrile or 1:1 acetonitrile:dichloromethane, the deprotection reagent was 3% trichloroacetic acid / dichloromethane, and the reaction time was 30 seconds with 4 deprotections. The activating agent used for the coupling reaction was 0.25 M 5-benzylthio tetrazole (BTT) in acetonitrile, and the reaction time was 160 seconds with 3 couplings. The 50 mM 1:1 acetonitrile:pyridine solution prepared with N,N-dimethyl-N’-(3-thio-3H-1,2,4- dithiazole-5-yl)formamidine was used to generate the phosphorothioate linkage, and the oxidation time was 120 seconds with 2 oxidations. The capping agent used was 10% acetic anhydride / tetrahydrofuran and N-methylimidazole / pyridine / tetrahydrofuran, and the capping time was 20 seconds with 2 cappings. All sequences were “DMT Off” at the end.

[0423] After the solid phase synthesis was complete, the solid supported oligonucleotide was treated with 500 pL of 3:1 ammonia water:ethanol at 45 °C for 16 h in a cryotube to cleave it from the solid support and remove additional labile protecting groups.

[0424] ​The oligonucleotides were precipitated with 10% v / v 5M NaCl solution and 3 volumes of absolute ethanol, centrifuged at 4°C, and the supernatant was removed. The oligonucleotides were re-dissolved in dd-H2O and purified by Agilent 1260 HPLC. The prepared solution was freeze-dried and then desalted by salt exchange. Then, LC-MS and UV spectroscopy were used for quantitative analysis. The microspectrophotometer OD 260 Determination of the concentration of the oligonucleotide solution.

[0425] The sense strands (KM001P-KM218P) and antisense strands (KM000G) were mixed in an equimolar ratio according to Table 1, incubated in a water bath at 95°C for 5 min, and then slowly returned to room temperature over 2-3 hours. The concentration and information of each double strand were confirmed, and then used for in vitro screening tests. The double-stranded siRNA numbers are shown in Table 2.

[0426] The sense strands (KM037P) and antisense strands (KM001G-KM512G) were mixed in an equimolar ratio according to Table 3, incubated in a water bath at 95°C for 5 min, and then slowly returned to room temperature over 2-3 hours. The concentration and information of each double strand were confirmed, and then used for in vitro screening tests. The double-stranded siRNA numbers are shown in Table 4.

[0427] Example 2: Synthesis of GalNAc derivatives

[0428] Compound 2 was also designed in the present application. The structure of the compound in this embodiment was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). The chemical shift δ is given in 10-6 (ppm) units. The NMR was measured by a Bruker nuclear magnetic instrument, and the measuring solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0429] LCMS was measured by: Agilent 1260 Infinity II (ESI) mass spectrometer, Waters UPLC H Class plus (ESI), or Shimadzu LCMS-2020 (ESI).

[0430] High-performance liquid chromatography (HPLC) analysis used Agilent 1260 or Shimadzu LC-20AD.

[0431] Preparative high-performance liquid chromatography (pre-HPLC) used GILSON GX-281 or Agilent 1260 Infinity II preparative liquid chromatography.

[0432] Chiral preparation uses critical fluid chromatography (SFC), and the instrument uses Shimadzu LC-30Adsf or Shimadzu LC-20AD.

[0433] Thin layer chromatography silica gel plate uses Anhui Liangchen Silicon Source Material Co., Ltd. GF254 acrylic adhesive silica gel plate. The silica gel plate used in thin layer chromatography (TLC) adopts a specification of 0.2 mm silica gel plate, and the specification of the silica gel plate used for thin layer chromatography separation and purification of the product is 0.5 mm.

[0434] Column chromatography generally uses 200-300 mesh silica gel from Anhui Liangchen Silicon Source Material Co., Ltd. as a carrier.

[0435] The determination of the average inhibition rate of the kinase and the IC50 value uses a SpectraMax i3X enzyme marker (MD, USA).

[0436] The known starting materials of the present embodiment can be synthesized according to methods known in the art or purchased from companies such as Bide Pharmaceutical, Leyan, Shaoyuan Chemical Technology, and Anning Chemicals.

[0437] Unless otherwise specified, the reactions are carried out under an argon atmosphere or a nitrogen atmosphere.

[0438] The argon atmosphere or the nitrogen atmosphere refers to that the reaction bottle is connected to an argon or nitrogen balloon with a volume of about 1 L.

[0439] The hydrogen atmosphere refers to that the reaction bottle is connected to a hydrogen balloon with a volume of about 1 L.

[0440] The hydrogenation reaction is usually vacuumed, filled with hydrogen, and repeated for 3 times.

[0441] The oxygen atmosphere refers to that the reaction bottle is connected to an oxygen balloon with a volume of about 1 L.

[0442] Unless otherwise specified, the solution refers to an aqueous solution, and the reaction temperature is room temperature, which is 20-30°C.

[0443] The monitoring of the reaction progress in the examples uses thin layer chromatography (TLC). The developing agent used in the reaction, the eluent system of column chromatography used for purifying compounds, and the developing agent system of thin layer chromatography include: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate system. The volume ratio of the solvents is adjusted according to the polarity of the compounds, and a small amount of triethylamine and acetic acid or other basic or acidic reagents can also be added for adjustment.

[0444] The following is the preparation of compound 2

[0445] First step

[0446] Dissolve 2a (5.00 g, 34.9 mmol) in 20 mL of ethanol, add ethyl 2-bromoacetate (5.83 g, 34.9 mmol), stir at 80 °C for 15 hours. Concentrate the reaction solution by distillation under reduced pressure, add 25 mL of isopropyl alcohol to the obtained residue, purify by recrystallization to obtain the title product 2b (7.34 g, yield: 91%) in the form of a yellow solid.

[0447] MS m / z (ESI): 230.1 [M+1].

[0448] Second step

[0449] Dissolve 2b (6.84 g, 29.7 mmol) in 50 mL of acetonitrile, protect under nitrogen, add 2-cyclopentenone (11.7 g, 142 mmol) and triethylamine (3.31 g, 32.6 mmol), stir at 25 °C for 24 hours under nitrogen protection. Add 50 mL of water to the reaction solution, extract the reaction solution with ethyl acetate (80 mL x 3), wash the organic phase with saturated sodium chloride solution (60 mL x 2), then dry over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 2c (5.81 g, yield: 61%) in the form of a yellow solid.

[0450] MS m / z (ESI): 312.1 [M+1].

[0451] Third step

[0452] Dissolve 2c (5.31 g, 16.5 mmol) in 60 mL of toluene, protect under nitrogen, add tributyltin hydride (11.9 g, 40.9 mmol) and azobisisobutyronitrile (543 mg, 3.31 mmol), react at 120 °C for 6 hours under nitrogen protection. Concentrate the reaction solution by distillation under reduced pressure, add 80 mL of ethyl acetate and hydrochloric acid (1 N, 40 mL) to the obtained residue, let the obtained liquid stand at 25 °C for 14 hours, suck off the organic phase, wash the remaining aqueous phase with ethyl acetate (100 mL x 3), obtain the crude title product 2d (3.27 g) in the form of a yellow liquid without purification, directly use the product in the next step reaction.

[0453] Fourth step

[0454] To 2d (3.27 g, 16.6 mmol) was added sodium bicarbonate solution (25 mL), acetonitrile (25 mL), 9-fluorenylmethyl-N-succinimidyl carbonate (6.71 g, 19.9 mmol), and the reaction was stirred at 25 °C for 2 h under nitrogen atmosphere. The reaction was extracted with ethyl acetate (80 mL x 3), and the organic phase was washed with saturated sodium chloride solution (100 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by silica gel column chromatography with the developing system B to give the title product 2e (2.01 g, yield: 27%) as a yellow oil.

[0455] 2e (2.01 g, 4.80 mmol) was separated by SFC (separation condition: column: DAICEL CHIRALPAK AD (250 mm x 30 mm, 10 pm); mobile phase: A-carbon dioxide: B-methanol, isocratic elution: B: 35%), to give single configuration compound 2e-1 and single configuration compound 2e-2.

[0456] 2e-1 (shorter retention time)

[0457] Yellow gum, 775 mg, yield: 39%. SFC analysis: retention time 1.830 min. (column: Chiralpak AD-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B: 5-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0458] MS m / z (ESI): 420.1 [M+1].

[0459] 2e-2 (longer retention time)

[0460] Yellow gum, 750 mg, yield: 37%. SFC analysis: retention time 2.193 min. (column: Chiralpak AD-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B: 5-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0461] MS m / z (ESI): 420.1 [M+1].

[0462] Fifth step

[0463] (R)-5,5-diphenyl-2-methyl-3,4-propanol-1,3,2-oxazaborolidine (79.3 mg, 286 pmol) was dissolved in 2 mL of toluene, protected by nitrogen, and cooled to 0 °C. Borane dimethyl sulfide complex (10 M, 171 pL) was added dropwise, and the reaction was allowed to proceed at 0 °C for 15 min. A toluene solution (8 mL) of 2e-1 (600 mg, 1.43 mmol) was then added, and the reaction was allowed to proceed at 0 °C for 30 min. 5 mL of methanol and 8 mL of water were added to the reaction at 0 °C, and the reaction was extracted with dichloromethane (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography with developing system A to give the title product 2f (500 mg, yield: 81%) as a yellow oil.

[0464] MS m / z (ESI): 422.0 [M + 1].

[0465] Sixth step

[0466] 2f (500 mg, 1.15 mmol) was dissolved in 10 mL of tetrahydrofuran, protected by nitrogen, and cooled to 0 °C. A tetrahydrofuran solution (2 M, 864 pL) of lithium borohydride was added dropwise, and the reaction was stirred at 25 °C for 1 h. 1 mL of ammonium chloride solution and 5 mL of water were added at 0 °C, and the reaction was extracted with dichloromethane (8 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The resulting residue was purified by silica gel column chromatography with developing system A to give the title product 2g (189 mg, yield: 63%) as a white solid.

[0467] MS m / z (ESI): 380.0 [M + 1].

[0468] Seventh step

[0469] 2g (25 mg, 64.57 pmol) was dissolved in 3 mL of dry pyridine, protected by nitrogen, and cooled to below 5 °C. A dichloromethane solution (5 mL) of 4,4'-dimethoxytrityl chloride (21.88 mg, 64.57 pmol) was then slowly added dropwise to the reaction, which was allowed to naturally rise to 25 °C and stirred for 12 h. The reaction was quenched by adding 1 mL of methanol, and the reaction was concentrated by distillation under reduced pressure. The resulting residue was purified by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-100%) to give the title product 2h (27 mg, yield: 58%) as a white solid.

[0470] Eighth step

[0471] Dissolution of 2h (26 mg, 38 pmol) and hexahydropyridine (6 mg, 73 pmol) in 3 mL of N,N-dimethylformamide, reaction at 25 °C for 12 hours, the reaction solution was purified by reverse phase liquid chromatography (mobile phase: A-water (0.05% ammonium bicarbonate), B-acetonitrile; gradient elution: B%: 10%-80%) without purification, to obtain the title product 2i (16 mg, yield: 91%) in the form of a white solid.

[0472] Ninth step

[0473] Dissolution of 2i (15 mg, 31.00 pmol) and dodecanedioic acid monomethyl ester (7.58 mg, 31.02 pmol) in 2 mL of dichloromethane, addition of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (14.15 mg, 37.21 pmol) and N,N-diisopropylethylamine (20.03 mg, 155.0 pmol), reaction at 25 °C for 2 hours. Concentration of the reaction solution by distillation under reduced pressure, the resulting residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 30%-100%), to obtain the title product 2j (14 mg, yield: 62%) in the form of a white solid.

[0474] Tenth step

[0475] Dissolution of 2j (12 mg, 16.62 pmol) in 2 mL of tetrahydrofuran and 2 mL of water, addition of lithium hydroxide (1.99 mg, 83.09 pmol), reaction at 25 °C for 12 hours. Concentration of the reaction solution by distillation under reduced pressure, the resulting residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 10%-70%), to obtain the title product 2k (10 mg, yield: 85%) in the form of a white solid.

[0476] Eleventh step

[0477] Dissolve 2k (5 mg, 7.07 μmol) and 1g (14.71 mg, 7.07 μmol) in 2 mL of dichloromethane, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.03 mg, 10.6 μmol) and N,N-diisopropylethylamine (4.57 mg, 35.36 μmol), and react at 25°C for 2 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 μm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 70%) to obtain the title product 2l (11 mg, yield: 55%) as a white solid.

[0478] Twelfth step

[0479] Dissolve 2l (10 mg, 3.94 μmol) in 3 mL of dichloromethane, add 4-dimethylaminopyridine (0.1 mg, 0.79 μmol), succinic anhydride (1.97 mg, 19.7 μmol), and N,N-diisopropylethylamine (5.09 mg, 39.4 μmol), and stir at 25°C for 15 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 μm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 10% - 90%) to obtain the title product 2 (4 mg, yield: 36%) as a beige solid.

[0480] MS m / z (ESI): 1162.9 [1 / 2 (M-301)].

[0481] 1H NMR (400 MHz, CD3OD) δ 7.39 - 7.36 (m, 2H), 7.30 - 7.28 (m, 1H), 7.27 - 7.24 (m, 5H), 7.21 - 7.18 (m, 1H), 6.87 - 6.82 (m, 4H), 5.33 - 5.31 (m, 3H), 5.08 - 5.03 (m, 4H), 4.57 - 4.54 (m, 3H), 4.17 - 4.04 (m, 12H), 4.02 - 3.97 (m, 4H), 3.89 - 3.84 (m, 4H), 3.78 - 3.77 (m, 6H), 3.70 - 3.65 (m, 12H), 3.56 - 3.50 (m, 4H), 3.48 - 3.46 (m, 1H), 3.33 - 3.31 (m, 2H), 3.15 - 3.11 (m, 12H), 2.46 - 2.42 (m, 8H), 2.25 - 2.21 (m, 6H), 2.13 - 2.11 (m, 9H), 2.02 - 2.00 (m, 9H), 1.94 - 1.91 (m, 18H), 1.71 - 1.49 (m, 22H), 1.48 - 1.42 (m, 2H), 1.31 - 1.25 (m, 12H), 0.50 - 0.46 (m, 12H).

[0482] Example 3: Design of modified siRNA

[0483] First, candidate oligonucleotide sequences complementary to human TTR sequences were generated using computer algorithm-based. The siRNA was designed as a double-stranded with 19 / 21 pairing of the sense strand and the antisense strand, and the antisense strand had 2 overhanging ends complementary to the mRNA sequence. The naked sequence of the siRNA oligonucleotide is listed in Table 1.

[0484] Table 1 Naked sequence of oligonucleotide

[0485] Based on SEQ ID NO: 1 and SEQ ID NO: 2, further modified nucleic acid sequences were designed, as shown in Tables 2 and 3. Among them, the sense strand number sequence KM037P and the antisense strand number sequence KM000G are the modification mode disclosed in WO2023241591A1, and the sense strand number sequence KM037P and the antisense strand number sequence KM512G are the modification mode disclosed in WO2019105418A1.

[0486] Taking KM037P000G as the positive control group, in order to further screen the modification type that the modification effect of the sense strand is better than KM037P and the modification of the antisense strand is better than KM000G, the sense strand and the antisense strand of the positive control group are combined with the antisense strand and the sense strand in this example, respectively. The combination number is shown in Tables 2 and 3.

[0487] Table 2 Modified sense strand sequence information

[0488] Table 3 Modified antisense strand sequence information

[0489] Note: Capital letters C, G, U, A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide; lowercase letter s represents that the linkage between the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate linkage.

[0490] Example 4: Preparation of siRNA conjugate

[0491] The preparation process of the oligonucleotide is as follows:

[0492] (1) Preparation of siRNA

[0493] The specific steps refer to Example 1.

[0494] (2) Preparation of ligand conjugated siRNA

[0495] The preparation of compound 2 is described in Example 2.

[0496] (3) Preparation of loading custom GalNAc solid support:

[0497] N,N-dimethylformamide to make a 8.1 mg / mL solution of N,N- diisopropylethylamine. 2-(7-Azabenzotriazol-1 -yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (2 eq) was weighed and dissolved in anhydrous acetonitrile to make a 14.6 mg / mL solution of 2-(7- azabenzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The GalNAc compound (Compound 1 -12 described above) was dissolved in the N,N-dimethylformamide solution of N,N- diisopropylethylamine (3 eq) followed by the addition of the acetonitrile solution of 2-(7-azabenzotriazol-1 -yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (2 eq). CPG (blank loading of 180 pmol / g, target loading of 40 pmol / g) was added to the mixture solution containing the GalNAc compound and mixed gently. The reaction was placed on a shaker (temperature: 25 °C, speed: 200 rpm) and shaken overnight. The reaction was added with dichloromethane to wash the CPG adhering to the wall of the bottle to the bottom of the bottle. The CPG was washed with dichloromethane for 3 times and anhydrous acetonitrile for 2 times, respectively. After each washing, the CPG was allowed to settle to the bottom of the bottle and the supernatant was carefully removed. The solid obtained after the last washing was dried under vacuum for 1 hour. 1 mg of 4-dimethylaminopyridine was weighed and dissolved in 1000 pL of anhydrous acetonitrile to make a 1 mg / mL solution of 4-dimethylaminopyridine in acetonitrile. 20 pL of N-methylimidazole was weighed and dissolved in 80 pL of anhydrous acetonitrile to make a Cap A solution. 40 pL of acetic anhydride was weighed and dissolved in 60 pL of anhydrous acetonitrile to make a Cap B1 solution. 60 pL of anhydrous pyridine was weighed and dissolved in 40 pL of anhydrous acetonitrile to make a Cap B2 solution. To the CPG support with the GalNAc compound, 65.4 pL of the 4-dimethylaminopyridine solution in acetonitrile, 12.95 pL of Cap A, 56.95 pL of Cap B1, and 6.33 pL of Cap B2 were added in sequence. The reaction was placed on a shaker (temperature: 25 °C, speed: 200 rpm) and shaken for 2 hours. The reaction was added with anhydrous acetonitrile to wash the CPG adhering to the wall of the bottle to the bottom of the bottle. The CPG was washed with anhydrous acetonitrile for 3 times. The solid was collected and dried under vacuum overnight to obtain the CPG with the GalNAc compound described in Example 2 (labeled as CPG-2) and the loading was determined.

[0498] The structure of CPG-2 is exemplarily shown as follows:

[0499] (4) Synthesis of siRNA with GalNAc coupled at 3' end of sense strand

[0500] After the loading of the custom GalNAc loaded solid support (i.e., compound CPG-2 prepared in step (3) above) is loaded into the empty column with the lower frit in place, the upper frit is placed on top. The nucleoside monomers are added one at a time in the order of the nucleotide sequence from 3' to 5'. Each addition of a nucleoside monomer includes four steps of deprotection, coupling, capping, and oxidation or sulfurization. The procedures are routine in the art.

[0501] Additional phosphoramidite monomers were obtained by commercial purchase, self-preparation, or custom synthesis. Phosphoramidite monomers were provided at 50 mM in acetonitrile or 1 : 1 acetonitrile: dichloromethane, deprotection reagent was 3% trichloroacetic acid in dichloromethane, and the reaction time was 30 seconds with 4 deprotections. The coupling reaction used 0.25 M 5-benzylthio tetrazole (BTT) in acetonitrile with a reaction time of 160 seconds and 3 couplings. The phosphorothioate linkage was generated using N,N-dimethyl-N'-(3-sulfenyl-3H-1,2,4- triazol-5-yl)formamidine at 50 mM in acetonitrile: pyridine (1 : 1) with an oxidation time of 120 seconds and 2 oxidations. The capping reagent used 10% acetic anhydride in tetrahydrofuran and N-methylimidazole / pyridine / tetrahydrofuran with a capping time of 20 seconds and 2 cappings. All sequences were "DMT Off" at the end.

[0502] After the solid phase synthesis is complete, the solid supported oligonucleotide is treated with 500 μL of ammonia: ethanol (3: 1) at 45 °C for 16 hours in a cryotube to cleave it from the solid support and subsequently remove additional labile protecting groups. Using compound CPG-2 as an example, after the solid phase synthesis described above to add the sequence and subsequent aminolysis, a portion of the functional groups are removed to become NGN-2.

[0503] The oligonucleotides were precipitated with 10% v / v 5 M sodium chloride solution and 3 volumes of absolute ethanol, centrifuged at 4 °C, and the supernatant was removed. The oligonucleotides were re-dissolved in dd-H2O and purified by Agilent 1260 HPLC. The prepared solution was freeze-dried and desalted by salt exchange. Then the quantitative analysis was performed by LC-MS and UV spectroscopy. The concentration of the oligonucleotide solution was determined by microspectrophotometer OD260. The sense and antisense strands were mixed in equimolar ratio, incubated at 95 °C water bath for 5 min, and then slowly recovered to room temperature within 2-3 hours. The concentration and information of each duplex were confirmed and then used for in vitro screening test. Referring to the above method, the compound 2 in Example 2 was conjugated with the sense strand of the oligonucleotide at the 3'-end through the linker to form the corresponding siRNA conjugate. Taking compound 2 as an example, the combination of exemplary conjugates is shown in Table 16.

[0504] Example 5: In vitro screening of siRNA

[0505] qPCR was used to detect the expression of siRNA containing various combinations of modifications on the target gene TTR of Hep3B cells.

[0506] 1. Cell culture and transfection

[0507] Hep3B cells (ATCC, Manassas, VA) were cultured in EMEM medium (ATCC, Cat# 30-2003) (supplemented with 10% FBS, streptomycin) at 37 °C in a 5% CO2 environment until the cells grew close to confluence, and then released from the plate by 0.25% trypsin EDTA solution (Shanghai Yonbiotech Co., Ltd., Cat# S310KJ). The cells were washed with PBS and resuspended at 0.25 x 10 6 cells / mL. During transfection, about 20,000 cells per well were seeded in a 96-well plate.

[0508] For Hep3B and primary cells, single-dose transfection was performed by adding 5 pL of Opti-MEM (Gibco, Cat# 31985-070) plus 0.5 pL of Lipofectamine RNAiMax (Invitrogen, Carlsbad Ca., Cat# 13778-150) per well to each well of a 96-well plate, and then adding 5 pL of each siRNA duplex to the mixture, which was incubated at room temperature for 5 minutes. Then 100 pL of complete medium containing the appropriate number of cells was added to the siRNA mixture. The cells were incubated for 24 hours before RNA extraction.

[0509] 2. Total RNA extraction and reverse transcription

[0510] Total RNA extraction from tissues / cells / blood using a magnetic bead method kit (Tiangen Biotech, catalog number: DP761) was performed at Thermo KingFisher. TM Total RNA was extracted and isolated using a Flex instrument. cDNA synthesis was performed using a cDNA reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number 4368813). Specifically: cells were harvested and lysed in 115 μL of lysis buffer at room temperature for 10 minutes. The lysed cells were added to deep-well plates pre-filled with a mixture of 10 μL magnetic beads and 100 μL isopropanol. The KingFisher instrument used a magnetic rod to capture the magnetic beads while continuously agitating them. The instrument then transferred the magnetic beads to 300 μL of protein-removing buffer RD per well and mixed for 5 minutes. The beads were then transferred to the next deep-well plate for DNase I digestion. After 10 minutes, 300 μL of protein-removing buffer RD was added for washing once, followed by two washes with 280 μL of rinsing buffer RW. Finally, the magnetic rod was used to capture the magnetic beads again, and RNA was eluted with 50 μL of RNase-free H2O at 60°C. Preparation of cDNA synthesis system: Add 2 μL of 5x HiScript III RT SuperMix (containing buffer, dNTPs, reverse transcriptase, RNase inhibitor, and random primers) and 8 μL of RNA extracted for in vitro screening as described above to each reaction tube. Use an ETC 811 PCR instrument (Beijing Dongsheng Innovation Technology Co., Ltd.) to generate cDNA through the following steps: 50℃ for 15 min, 85℃ for 5 s, and hold at 4℃.

[0511] 3. Real-time quantitative PCR

[0512] The industry-recognized modification combination mode KM037P000 was used as a positive reference modification combination. In each well of a 96-well plate (Applied Biosystems, item number 4483354), 4 μL of cDNA was added to a premix solution containing: 5 μL AceQ Universal Probe Mix (Nanjing Novozyme BioTech Co., Ltd., item number Q513), 0.1 μL GAPDH TaqMan probe (Suzhou Jindunzhi Biotechnology Co., Ltd.), 0.1 μL target TaqMan probe (Suzhou Jindunzhi Biotechnology Co., Ltd.), and 0.2 μL of GAPDH-F / R, target-F / R primers. The primers and probes used are shown in Table 4 for human primers and probe information. Real-time fluorescence quantitative PCR was detected on a Biosystems Quant Studio 5 system (Applied Biosystems). Each sample was subjected to at least two technical repeats. Real-time data were analyzed using the ΔΔCt method to calculate the retention rate of target gene expression levels. A corresponding reference was set for each test to eliminate errors in different batches of tests. Finally, the results of each test were divided by the reference to obtain Tables 5 and 6.

[0513] Table 4 Primer and probe information for real-time fluorescence quantitative PCR

[0514] Note: h represents human, m represents mouse, F represents forward primer, R represents reverse primer, P represents probe, 6-FAM and BHQ1 represent fluorescent groups and quenching groups, respectively.

[0515] Table 5 Fluorescence quantitative PCR results of sense strand modification screening

[0516] The combinations that were better than the reference modification at both 1 nM and 4 nM were selected for further evaluation (in Table 8, the modification combinations with both 1 nM and 4 nM less than 1).

[0517] Table 6 Fluorescence quantitative PCR results of antisense strand modification screening

[0518] Based on the results of the fluorescent quantitative PCR test in Table 5 and Table 6, the modification combination of the sense strand superior to KM037P and the antisense strand superior to KM000G (about 20%) was selected for combination, and the sequence and combination form are shown in Table 7. On this basis, IC50determination was carried out, and 7 different concentrations were set as follows: 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, 0.00064 nM (5-fold dilution ratio), and the final result was taken as siRNA concentration as the horizontal coordinate and the retention rate of target gene expression level as the vertical coordinate. Graphpad prism software was used for log(inhibitor)vs.response--Variable slope(four parameters)parameter fitting, and IC50was calculated.

[0519] Table 7 Numbering information of double-stranded human TTR for cross-combination screening of sense strand and antisense strand

[0520] The siRNA in Table 7 was subjected to IC50determination, and the determination results are shown in Figures 1A-1F.

[0521] Based on the IC50data, most of the candidate modification types are superior to the positive control (KM037P512G) to varying degrees, specifically: KM037P198G, KM037P301G, KM058P205G, KM072P205G, KM072P258G, KM072P395G, KM072P509G, KM084P506G, KM092P491G, KM112P198G, KM113P084G, KM113P099G, KM113P334G, KM114P204G, KM114P205G, KM114P209G, KM114P488G, KM114P491G, KM123P258G, KM179P265G, KM200P205G, KM200P258G, KM200P301G, KM200P506G, KM206P174G, KM206P197G, KM217P198G. In addition, it was found that the half-inhibitory concentration of the positive control KM037P000G was about half of KM037P512G, which exhibited better activity in vitro, and therefore KM037P000G was used as the positive control in the subsequent examples.

[0522] Example 6: Verification of the universality of the modification mode of siRNA on different sequences

[0523] To verify the universality of the candidate modification on different sequences, and to verify the activity of the candidate modification on the remaining four product sequences disclosed in the prior art patents (including: ALN_APP (PCT Publication No: WO2020132227), Cemdisiran-C5 (PCT Publication No: WO2014160129), Zilebesiran-AGT (PCT Publication No: WO2016196111) and APOC3 (PCT Publication No: WO2019128611)), based on the results of human TTR sequence, different modified siRNAs were synthesized according to Example 1, and after annealing, the experimental steps in Example 3 were referred to, and IC50determination was performed on Hep3B. The sequence information is shown in Table 8 and Table 9, and the test results are shown in Figures 2A-2R.

[0524] Table 8: Single-stranded modification information table corresponding to four human sequences (APP / C5 / AGT / APOC3)

[0525] Table 9: Double-stranded pairing information table corresponding to four sequences (APP / C5 / AGT / APOC3)

[0526] As can be seen from Table 10 and Figures 2A-2R, the experimental results demonstrate that siRNAs containing various modification modes such as KM072P205G and KM114P205G all have good in vitro inhibition effect.

[0527] Table 10: IC50of cross combination of sense and antisense strands at C5 target 50 Screening results

[0528] Note: ++ indicates that IC50is less than or equal to 0.1, + indicates that IC50value is greater than 0.1 and less than 0.2; A indicates that the maximum inhibition rate is greater than 80%.

[0529] Example 7: Evaluation of plasma stability

[0530] The in vitro plasma stability of different modifications was evaluated using mixed gender C57BL / 6 mouse plasma (Beijing Huizhi Heyuan Biotechnology Co., Ltd., Cat No. 032E21.21 / 032E22.22, female and male mixed at a ratio of 1:1), mixed gender SD rat plasma (Beijing Huizhi Heyuan Biotechnology Co., Ltd., 033D11.21 / 033D12.21, female and male mixed at a ratio of 1:1), mixed human plasma (Beijing Huizhi Heyuan Biotechnology Co., Ltd., 0191A1.11), and mixed cynomolgus monkey plasma (Beijing Huizhi Heyuan Biotechnology Co., Ltd., 032B11.21 / 032B12.21, female and male mixed at a ratio of 1:1). The siRNAs were incubated at a concentration of 2 μΜ at 37°C for 0 hours, 2 hours, 8 hours, and 24 hours, respectively. The siRNAs were recovered by solid-phase extraction and analyzed by triple quadrupole mass spectrometry. The results of each group were normalized to 0 hours. The results are shown in Tables 11-14.

[0531] As shown in Tables 11-14, the results showed that, relative to the reference (KM037P000G) modification, the remaining modifications had good 24-hour plasma stability in any species, except for KM113P084G and KM113P099G.

[0532] Table 11. Modified siRNA mixed gender and donor human plasma stability results (antisense strand)

[0533] Table 12. Modified siRNA mixed gender and donor cynomolgus monkey plasma stability results (antisense strand)

[0534] Table 13. Modified siRNA mixed gender and donor C57BL / 6 mouse plasma stability results (antisense strand)

[0535] Table 14. Mixed SD rat gender and donor plasma stability results (antisense strand)

[0536] Example 8: In vivo verification of preferred siRNA modifications

[0537] KM113P099G showed better activity in vitro, therefore the antisense strand of KM113P099G was modified to enhance its stability, specifically, the 2'-F modification at the 20th position (5'-3') of the antisense strand was replaced by 2'-oMe modification to increase its plasma stability in vivo (KM099G corresponds to the antisense strand 20th position without 2'-F modification corresponds to KM025G in Table 3), then the single-stranded siRNA of Table 15 was synthesized, and annealed into double-stranded according to Table 16. C57BL / 6 mice (female, 18-21 g, 6-8 weeks) were randomly grouped according to Figure 3, 4 animals per group, and the dosage was calculated according to the body weight of each animal, and a single dose was given by subcutaneous injection. The siRNA compound was configured at 0.1 mg / mL stock solution (0.9% sodium chloride injection solution as solvent), and the siRNA compound was dissolved with 0.9% sodium chloride injection solution before the experiment and made up to the required solution concentration and volume. The dosing volume of normal saline and siRNA compound was 5 mL / kg. Blood was collected from the orbital venous plexus of the mice before administration (recorded as day 0), 7, 14, 21, and 28 days after administration. At each time point, the expression level of TTR protein in mouse serum was detected by ELISA kit (Abeam, ab282297), and after reverse transcription, the TTR mRNA level in mouse liver was detected by real-time fluorescence quantitative PCR (qPCR) according to Example 3 (see Table 6 for primer and probe information).

[0538] The ELISA detection results and qPCR detection results are shown in Table 17 and Figures 3A-3B.

[0539] The results show that the inhibitory activity of L96-mTTR072P205G, L96-mTTR114P205G, and L96-mTTR113P025G of the three modification types is significantly better than that of the control group L96-mTTR037P000G, and the maximum knockdown efficiency in 28 days is also significantly better than that of the control group. Similarly, the three modification types of compound 2-mTTR072P205G, compound 2-mTTR114P205G, and compound 2-mTTR113P025G are also significantly better than the control groups L96-mTTR037P000G and compound 2-mTTR037P000G, and the maximum knockdown efficiency is also significantly better than that of the control group.

[0540] Table 15: Single-stranded modification information table of mouse TTR sequence corresponding modification

[0541] Table 16: Double-stranded pairing information table of mouse TTR

[0542] Table 17 In vivo screening ELISA quantitative information of different modifications

[0543] Example 9: Liver S9 fraction stability evaluation of candidate modifications

[0544] Based on the results of Example 5, candidate KM072P205G, KM114P205G, KM113P025G were evaluated with human liver S9 fraction (Huzhou and Source Biotech (Suzhou) Co., Ltd., Catalog No: 0131A1.03), which is rich in various enzymes and can simulate the in vivo environment of siRNA. Specifically, different modified siRNAs at a concentration of 1 mM were incubated with 1.0 mg / mL liver S9 for 0 hours, 2 hours, 4 hours, 8 hours and 24 hours, respectively, and then the siRNAs were recovered by liquid phase extraction and analyzed by liquid chromatography-mass spectrometry. The results of each group were normalized to 0 hours, and the results are shown in Tables 18-19.

[0545] The results show that KM072P205G, KM113P025G and KM114P205G have better human liver S9 fraction stability compared to the reference.

[0546] Table 18 Modification siRNA mixed gender and donor human liver S9 stability results (antisense strand)

[0547] Table 19 Modification siRNA mixed gender and donor human liver S9 stability results (sense strand)

[0548] Example 10: In vitro activity data of KM113P025G

[0549] To verify the in vitro activity of KM113P025G, the reference examples 3, 5 and 6 were used to verify the activity of the candidate modification on five product sequences at the same time (including: TTR (sense and antisense strands as shown in SEQ ID NO: 1 and SEQ ID NO: 2), ALN_APP (PCT Publication No: WO2020132227), Cemdisiran-C5 (PCT Publication No: WO2014160129), Zilebesiran-AGT (PCT Publication No: WO2016196111) and APOC3 (PCT Publication No: WO2019128611)), based on Example 1 to synthesize different modified siRNAs, after annealing, the experimental steps in Examples 3, 5 and 6 were used to determine the IC50 on Hep3B. The test results are shown in Figures 4A-4E.

[0550] As can be seen from FIGS. 4A-4E, the in vitro activity of KM113P025G is significantly better than the positive control KM037P000G.

[0551] Example 11: In vivo verification of preferred siRNA modification in humanized mice

[0552] To further verify the in vivo activity of the modified siRNA compounds, different modification modes of compounds were synthesized, as shown in Tables 20 and 21, and B-hMASP2 mice (female, 18-21 g, 6-8 weeks, item number: 110872, Bao Sesitu (Beijing) Pharmaceutical Technology Co., Ltd.) were used, with 4 animals in each group. Before administration, the serum MASP2 protein expression level of the mice was detected by an ELISA kit (Abeam, ab278121), and the snakes were grouped according to the expression amount. The compound administration amount in Table 21 was calculated according to the body weight of each animal, and subcutaneous injection was used for single administration, with a dose of 3 mg / kg. Blood was collected from the orbital venous plexus of the mice before administration (recorded as day 0), and on days 5, 14, 21, 30, and 42 after administration. At each time point, the serum MASP2 protein expression level of the mice was detected by an ELISA kit (Abeam, ab278121).

[0553] The ELISA detection results are shown in Table 22, and the results show that KM072P205G and KM114P205 exhibit good activity.

[0554] Table 20: Single-stranded modification information table corresponding to human MASP2 sequence modification

[0555] Table 21: Double-stranded pairing information table corresponding to human MASP2

[0556] Table 22: ELISA quantitative information of in vivo screening of siRNA targeting MASP2 with different modifications

[0557] Example 12: Pharmacokinetics (PK) of modified siRNA molecules in SD rats

[0558] To verify the pharmacokinetic properties of the modified siRNA compounds in vivo, the compounds of preferred siRNA modification mode were synthesized using GL6 and compound 2 in WO2023143571A1 as GalNAc ligands, respectively (see Table 23). SD female rats were used, 9 rats in each group, and administered subcutaneously at a single dose of 10 mg / kg. Blood plasma (100 ul) was collected at 5 min, 30 min, 60 min and 240 min before and after administration, and placed in a storage tube containing EDTA anticoagulant. In addition, the livers of 3 rats in each group were taken at 4 hours, 7 days and 14 days after administration, respectively, and the contents of siRNA sense strand (SS) and antisense strand (AS) were detected by HPLC-mass spectrometry. The results showed (see Tables 24 and 25) that the compound modified with KM114P205G had a higher exposure under the same dose and administration method.

[0559] Table 23 Compound information used in this example

[0560] Table 24 SD rat plasma PK results

[0561] Table 25 SD rat liver PK results

[0562] Example 13: Toxicity test of modified siRNA molecules in SD rats

[0563] To verify the safety of the modified siRNA compounds in vivo, SD rat toxicity tests were conducted using the compounds shown in Table 23, a total of three groups, four SD rats (6-8 weeks old, half male and half female) in each group, administered subcutaneously at 300 mg / kg on D0 and D7, a total of two times, and blood biochemical tests were performed 24 hours after the last administration. The results showed that there were no changes in animal body weight and food intake in each administration group, and no abnormalities were found in gross anatomy. The results of liver and kidney function showed (see Table 26) that the safety of the two compounds was equivalent.

[0564] Table 26 SD rat results

[0565] Each technical feature of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, however, as long as the combinations of the technical features do not exist contradictory, they should be considered as the scope of the present disclosure.

[0566] The above-described embodiments are merely illustrative of several embodiments of the present disclosure, which are described in a more specific and detailed manner, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present disclosure, and these are all within the scope of the present disclosure. Therefore, the scope of protection of the patent of the present disclosure should be subject to the appended claims.

Claims

1. A double-stranded oligonucleotide that inhibits expression of a target gene, the double-stranded oligonucleotide comprising a sense strand and an antisense strand that is complementary to at least a portion of an mRNA of the target gene, wherein, each of the sense strand and the antisense strand is independently 15-30 nucleotides in length, the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region, each nucleotide of the sense strand and the antisense strand is a modified nucleotide, and the double-stranded oligonucleotide comprises at least one modified internucleotide linkage, the modified nucleotide comprises at least a 2'-methoxy modified nucleotide or a 2'-fluoro modified nucleotide, the antisense strand of the double-stranded oligonucleotide comprises at least one of the following modifications: (1) at least 4 2'-fluoro modified nucleotides; (2) at least 8 2'-methoxy modified nucleotides; (3) 2-4 modified internucleotide linkages; and / or; the sense strand of the double-stranded oligonucleotide comprises at least one of the following modifications: (1) at least 1 2'-fluoro modified nucleotide; (2) at least 11 2'-methoxy modified nucleotides; (3) 2-4 modified internucleotide linkages.

2. The double-stranded oligonucleotide of claim 1, wherein, the sense strand and the antisense strand are substantially reverse-complementary, substantially fully reverse-complementary, or fully reverse-complementary.

3. The double-stranded oligonucleotide of claim 1 or 2, wherein, the sense strand and the antisense strand are 15-25 nucleotides in length, preferably 17-21 nucleotides in length, more preferably 19-21 nucleotides in length.

4. The double-stranded oligonucleotide of claim 3, wherein, the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length.

5. The double-stranded oligonucleotide of any one of claims 1-4, wherein, the modified internucleotide linkage is a phosphorothioate linkage.

6. The double-stranded oligonucleotide of any one of claims 1-5, wherein, the antisense strand of the double-stranded oligonucleotide comprises at least 4 2'-fluoro modified nucleotides, at least 8 2'-methoxy modified nucleotides; Preferably, the antisense strand of the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

7. The double-stranded oligonucleotide of claim 6, wherein, at least 3 of the nucleotides at positions 2, 6, 14, 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides; Preferably, the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are 2'-fluoro modified nucleotides.

8. The double-stranded oligonucleotide of claim 6 or 7, wherein, at least 7 of the nucleotides at positions 1, 9, 11, 13, 15, 17, 19, 21 from the 5' end of the antisense strand are 2'-methoxy modified nucleotides; Preferably, the nucleotides at positions 1, 9, 11, 13, 15, 17, 19, and 21 from the 5' end of the antisense strand are 2'-methoxy modified nucleotides.

9. The double-stranded oligonucleotide of claim 7 or 8, wherein, the nucleotides at positions 3, 4, 5, 6, 7, 8 from the 5' end of the antisense strand comprise at least 2, 3, 4, or 5 consecutive 2'-fluoro modified nucleotides.

10. The double-stranded oligonucleotide of any one of claims 6-9, wherein, the phosphorothioate linkages are present between: (1) the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 5' end of the antisense strand; and / or (2) the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 3' end of the antisense strand; Preferably, the phosphorothioate linkages are present between the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 5' end of the antisense strand, and the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides from the 3' end of the antisense strand.

11. The double-stranded oligonucleotide of any one of claims 6-10, wherein, The phosphorothioate linkage is present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

12. The double-stranded oligonucleotide of any one of claims 1-11, wherein, The antisense strand of the double-stranded oligonucleotide comprises 6 2'-fluoro-modified nucleotides and 15 2'-methoxy-modified nucleotides; Preferably, the antisense strand of the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

13. The double-stranded oligonucleotide of claim 12, wherein, The nucleotides at positions 2, 5, 6, 7, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, the phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

14. The double stranded oligonucleotide of any one of claims 1-11, wherein, The antisense strand of the double-stranded oligonucleotide comprises 7 2'-fluoro-modified nucleotides and 14 2'-methoxy-modified nucleotides; Preferably, the antisense strand of the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

15. The double-stranded oligonucleotide of claim 14, wherein, The antisense strand is selected from any one of the following combinations: (1) the nucleotides at positions 2, 4, 6, 7, 14, 16, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or (2) the nucleotides at positions 2, 5, 6, 7, 14, 16, 20 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, the phosphorothioate linkages are present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand of any one of the combinations.

16. The double stranded oligonucleotide of any one of claims 1-11, wherein, The antisense strand of the double-stranded oligonucleotide comprises 8 2'-fluoro-modified nucleotides and 13 2'-methoxy-modified nucleotides; Preferably, the antisense strand of the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

17. The double-stranded oligonucleotide of claim 16, wherein, The antisense strand is selected from any one of the following combinations: (1) the nucleotides at positions 2, 3, 4, 5, 6, 8, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (2) the nucleotides at positions 2, 4, 5, 6, 12, 14, 16, 18 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (3) the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (4) the 2nd, 4th, 6th, 7th, 8th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (5) the 2nd, 4th, 6th, 7th, 12th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (6) the 2nd, 3rd, 6th, 7th, 12th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or (7) the 2nd, 4th, 5th, 6th, 10th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are phosphorothioate linkages between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand in any of the above groups.

18. The double stranded oligonucleotide of any one of claims 1-11, wherein, the antisense strand of the double-stranded oligonucleotide comprises 9 2'-fluoro-modified nucleotides and 12 2'-methoxy-modified nucleotides; Preferably, the antisense strand of the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

19. The double-stranded oligonucleotide of claim 18, wherein, the antisense strand is any of the following groups: (1) the 2nd, 3rd, 5th, 6th, 8th, 10th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (2) the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (3) the 2nd, 4th, 5th, 6th, 7th, 12th, 14th, 16th, and 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or (4) the 2nd, 3rd, 4th, 5th, 6th, 10th, 12th, 14th, and 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are phosphorothioate linkages between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand in any of the above groups.

20. The double-stranded oligonucleotide of any one of claims 1-11, wherein, the antisense strand of the double-stranded oligonucleotide comprises 10 2'-fluoro-modified nucleotides and 11 2'-methoxy-modified nucleotides; Preferably, the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

21. The double-stranded oligonucleotide of claim 20, wherein, the 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 14th, 16th, and 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides, Preferably, there are phosphorothioate linkages between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

22. The double-stranded oligonucleotide of any one of claims 1-11, wherein, The antisense strand of the double-stranded oligonucleotide comprises 11 2'-fluoro-modified nucleotides and 10 2'-methoxy-modified nucleotides; Preferably, the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

23. The double-stranded oligonucleotide of claim 22, wherein, The antisense strand is selected from any one of the following combinations: (1) the 2nd, 3rd, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (2) the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are phosphorothioate linkages between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

24. The double-stranded oligonucleotide of any one of claims 1-11, wherein, The antisense strand of the double-stranded oligonucleotide comprises 11 2'-fluoro-modified nucleotides and 10 2'-methoxy-modified nucleotides; Preferably, the double-stranded oligonucleotide further comprises 4 phosphorothioate linkages.

25. The double-stranded oligonucleotide of claim 24, wherein, The antisense strand is selected from any one of the following combinations: (1) the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (2) the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are phosphorothioate linkages between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

26. The double-stranded oligonucleotide of any one of claims 1-11, wherein, The antisense strand is selected from any one of the following combinations: (1) the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (2) the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (3) the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (4) the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides. (4) the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides. Preferably, the phosphorothioate linkage is present between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

27. The double-stranded oligonucleotide of claim 26, wherein, The antisense strand is any one selected from the following combinations: (1) the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (2) the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, the phosphorothioate linkage is present between the 1st and 2nd, between the 2nd and 3rd, between the 19th and 20th, and between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

28. The double-stranded oligonucleotide of any one of claims 1-27, wherein, The sense strand of the double-stranded oligonucleotide comprises at least 1 2'-fluoro-modified nucleotide, and at least 11 2'-methoxy-modified nucleotides; Preferably, the sense strand of the double-stranded oligonucleotide further comprises 2 or 4 phosphorothioate linkages.

29. The double-stranded oligonucleotide of claim 28, wherein, The 9th nucleotide from the 5' terminus of the sense strand is a 2'-fluoro-modified nucleotide.

30. The double-stranded oligonucleotide of claim 28 or 29, wherein, At least 10 of the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, 19th nucleotides from the 5' terminus of the sense strand are 2'-methoxy-modified nucleotides; Preferably, the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, 19th nucleotides from the 5' terminus of the sense strand are 2'-methoxy-modified nucleotides.

31. The double-stranded oligonucleotide of claim 29 or 30, wherein, At least 2 of the 7th, 8th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and at least 1 of the 5th, 6th, 10th, 11th, 12th nucleotides is a 2'-fluoro-modified nucleotide.

32. The double-stranded oligonucleotide of any one of claims 28-31, wherein, The phosphorothioate linkage is present between: (1) the 1st and 2nd, between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; and / or (2) the 1st and 2nd, between the 2nd and 3rd nucleotides from the 3' terminus of the sense strand; Preferably, the phosphorothioate linkage is present between the 1st and 2nd, between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand.

33. The double-stranded oligonucleotide of any one of claims 28-32, wherein, The sense strand of the double-stranded oligonucleotide comprises 3 2'-fluoro-modified nucleotides, and 16 2'-methoxy-modified nucleotides; Preferably, the double-stranded oligonucleotide further comprises 2 phosphorothioate linkages.

34. The double-stranded oligonucleotide of claim 33, wherein, the nucleotides at positions 6, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand in any of the groups.

35. The double-stranded oligonucleotide of any one of claims 28-32, wherein, the sense strand of the double-stranded oligonucleotide comprises 4 2'-fluoro-modified nucleotides, 15 2'-methoxy-modified nucleotides; Preferably, the double-stranded oligonucleotide further comprises 2 phosphorothioate linkages.

36. The double-stranded oligonucleotide of claim 35, wherein, the sense strand is selected from any of the following groups: (1) the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (1) the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (1) the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (1) the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (1) the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (1) the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (1) the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand in any of the groups. the sense strand of the double-stranded oligonucleotide comprises 4 2'-fluoro-modified nucleotides, 15 2'-methoxy-modified nucleotides; 37. The double-stranded oligonucleotide of any one of claims 28-32, wherein, Preferably, the double-stranded oligonucleotide further comprises 2 phosphorothioate linkages. the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; 38. The double-stranded oligonucleotide of claim 37, wherein, Preferably, there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand in any of the groups. the sense strand of the double-stranded oligonucleotide comprises 4 2'-fluoro-modified nucleotides, 15 2'-methoxy-modified nucleotides; 39. The double-stranded oligonucleotide of any one of claims 28-32, wherein, Preferably, the double-stranded oligonucleotide further comprises 2 phosphorothioate linkages. the nucleotides at positions 5, 6, 7, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand in any of the groups. the sense strand of the double-stranded oligonucleotide comprises 4 2'-fluoro-modified nucleotides, 15 2'-methoxy-modified nucleotides; Preferably, the double-stranded oligonucleotide further comprises 2 phosphorothioate linkages.

40. The double-stranded oligonucleotide of claim 39, wherein, The sense strand is selected from any one of the following combinations: (1) the nucleotides at positions 5, 7, 8, 9, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (2) the nucleotides at positions 6, 7, 8, 9, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are also phosphorothioate bonds between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand in any one of the combinations.

41. The double-stranded oligonucleotide of any one of claims 28-32, wherein, The sense strand of the double-stranded oligonucleotide comprises 7 2'-fluoro-modified nucleotides and 12 2'-methoxy-modified nucleotides; Preferably, the double-stranded oligonucleotide further comprises 2 phosphorothioate bonds.

42. The double-stranded oligonucleotide of claim 41, wherein, The nucleotides at positions 5, 7, 8, 9, 10, 11, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are also phosphorothioate bonds between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

43. The double-stranded oligonucleotide of any one of claims 1-32, wherein, The sense strand is selected from any one of the following combinations: (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (2) the nucleotides at positions 7, 8, 9, 11 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (3) the nucleotides at positions 7, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (4) the nucleotides at positions 7, 8, 9, 10 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (5) the nucleotides at positions 8, 9, 10, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are also phosphorothioate bonds between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

44. The double-stranded oligonucleotide of claim 43, wherein, The sense strand is selected from any one of the following combinations: (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (2) the nucleotides at positions 7, 8, 9, 11 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (3) the nucleotides at positions 7, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

45. The double-stranded oligonucleotide of any one of claims 1-11, 28-32, wherein, Preferably, the nucleotide at the 9th position from the 5' end of the sense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th positions are 2'-methoxy-modified nucleotides. Preferably, the nucleotide at the 9th position from the 5' end of the sense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th positions are 2'-methoxy-modified nucleotides. Preferably, the nucleotide at the 9th position from the 5' end of the sense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th positions are 2'-methoxy-modified nucleotides. Preferably, the nucleotide at the 9th position from the 5' end of the sense strand is a 2'-fluoro-modified nucleotide, and the nucleotides at the 1st, 2nd, 3rd, 4th, 13th, 14th, 15th, 16th, 17th, 18th, and 19th positions are 2'-methoxy-modified nucleotides. Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand. Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

46. The double-stranded oligonucleotide of claim 45, wherein, Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand. Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand. Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand. Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand. Preferably, there is a phosphorothioate bond between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand.

47. The double-stranded oligonucleotide of claim 45 or 46, wherein, the nucleotides at positions 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and one and only one of the nucleotides at positions 5, 11 or 12 is a 2'-fluoro-modified nucleotide; the nucleotides at positions 2, 6, 7, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and one and only one of the nucleotides at positions 4 or 5 is a 2'-fluoro-modified nucleotide; Preferably, there are also phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand. Preferably, there are also phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

48. The double-stranded oligonucleotide of claim 47, wherein, the sense strand is selected from any one of the following combinations: (1) the nucleotides at positions 5, 7, 8, 9 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; (2) the nucleotides at positions 7, 8, 9, 11 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or (3) the nucleotides at positions 7, 8, 9, 12 from the 5' end of the sense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; the antisense strand is selected from any one of the following combinations: (1) the nucleotides at positions 2, 4, 6, 7, 10, 12, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; or (2) the nucleotides at positions 2, 5, 6, 7, 14, 16 from the 5' end of the antisense strand are 2'-fluoro-modified nucleotides, and the remaining nucleotides are 2'-methoxy-modified nucleotides; Preferably, there are also phosphorothioate linkages between the 1st and 2nd nucleotides, and between the 2nd and 3rd nucleotides from the 5' end of the sense strand. Preferably, there are also phosphorothioate linkages between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, and between the 20th and 21st nucleotides from the 5' end of the antisense strand.

49. The double-stranded oligonucleotide of any one of claims 1-11, 28-32, 45-47, wherein, the double-stranded oligonucleotide is selected from any one of the following combinations: (1) the 5th, 7th, 8th, 9th nucleotides from the 5' terminal end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminal end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (2) the 7th, 8th, 9th, 12th nucleotides from the 5' terminal end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminal end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (3) the 7th, 8th, 9th, 11th nucleotides from the 5' terminal end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminal end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (4) the 6th, 8th, 9th nucleotides from the 5' terminal end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 8th, 14th, 16th nucleotides from the 5' terminal end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (5) the 7th, 8th, 9th nucleotides from the 5' terminal end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminal end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (6) the 7th, 8th, 9th nucleotides from the 5' terminal end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 8th, 10th, 12th, 14th, 16th nucleotides from the 5' terminal end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (7) the 5th, 6th, 7th, 9th nucleotides from the 5' terminal end of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminal end of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (8) the 5th, 6th, 7th, 9th nucleotides from the 5' terminal of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminal of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (9) the 5th, 7th, 8th, 9th nucleotides from the 5' terminal of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th nucleotides from the 5' terminal of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (10) the 5th, 7th, 8th, 9th nucleotides from the 5' terminal of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 8th, 12th, 14th, 16th, 18th nucleotides from the 5' terminal of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (11) the 5th, 7th, 8th, 9th nucleotides from the 5' terminal of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminal of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (12) the 5th, 8th, 9th, 12th nucleotides from the 5' terminal of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminal of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (13) the 6th, 7th, 8th, 9th nucleotides from the 5' terminal of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminal of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (14) the 7th, 8th, 9th, 10th nucleotides from the 5' terminal of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminal of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (15) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (16) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (17) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 7th, 12th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (18) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 8th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (19) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 12th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (20) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 12th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (21) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (22) the nucleotides at the 8th, 9th, 10th, 12th position from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th position from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (23) the nucleotides at the 7th, 8th, 9th, 11th, 12th position from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at the 2nd, 3rd, 4th, 5th, 6th, 10th, 12th, 14th, 16th position from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (24) the nucleotides at the 5th, 7th, 8th, 9th, 11th, 12th position from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th position from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (25) the nucleotides at the 5th, 7th, 8th, 9th, 11th, 12th position from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th position from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (26) the nucleotides at the 5th, 7th, 8th, 9th, 11th, 12th position from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at the 2nd, 3rd, 5th, 6th, 8th, 10th, 12th, 14th, 16th position from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (27) the nucleotides at the 5th, 7th, 8th, 9th, 11th, 12th position from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at the 2nd, 3rd, 4th, 5th, 6th, 7th, 10th, 12th, 14th, 16th, 18th, 20th position from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (28) the nucleotides at the 6th, 7th, 8th, 9th, 11th, 12th position from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the nucleotides at the 2nd, 3rd, 6th, 7th, 12th, 14th, 16th, 20th position from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (29) the 6th, 7th, 8th, 9th, 11th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 10th, 14th, 16th, 20th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (30) the 5th, 7th, 8th, 9th, 10th, 11th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, in any one of the groups, there are phosphorothioate linkages between the 1st and 2nd, 2nd and 3rd nucleotides from the 5' terminus of the sense strand. Preferably, in any one of the groups, there are phosphorothioate linkages between the 1st and 2nd, 2nd and 3rd, 19th and 20th, 20th and 21st nucleotides from the 5' terminus of the antisense strand.

50. The double-stranded oligonucleotide of claim 49, wherein, The double-stranded oligonucleotide is any one selected from the following groups: (1) the 5th, 7th, 8th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (2) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (3) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (4) the 7th, 8th, 9th, 10th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 5th, 6th, 12th, 14th, 16th, 18th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (5) the 8th, 9th, 10th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 3rd, 4th, 5th, 6th, 7th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, in any one of the groups, there are phosphorothioate bonds between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; Preferably, in any one of the groups, there are phosphorothioate bonds between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

51. The double-stranded oligonucleotide of claim 50, wherein, The double-stranded oligonucleotide is any one selected from the following groups: (1) the 5th, 7th, 8th, 9th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; (2) the 7th, 8th, 9th, 12th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 4th, 6th, 7th, 10th, 12th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; or (3) the 7th, 8th, 9th, 11th nucleotides from the 5' terminus of the sense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; the 2nd, 5th, 6th, 7th, 14th, 16th nucleotides from the 5' terminus of the antisense strand are 2'-fluoro-modified nucleotides, and the rest of the nucleotides are 2'-methoxy-modified nucleotides; Preferably, in any one of the groups, there are phosphorothioate bonds between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides from the 5' terminus of the sense strand; Preferably, in any one of the groups, there are phosphorothioate bonds between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 19th and 20th nucleotides, between the 20th and 21st nucleotides from the 5' terminus of the antisense strand.

52. The double-stranded oligonucleotide of any one of claims 1-51, wherein, the modified nucleotides further comprise one or more nucleotide modifications selected from the group consisting of: 2'-deoxynucleotides, 2',3'-seco nucleotide mimics, locked nucleotides (LNA), unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), bicyclic nucleic acids (BNA), 2'-F- arabinonucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribothymidines, inverted nucleotides, inverted abasic nucleotides (Invab), inverted 2'-OMe nucleotides, inverted 2'-deoxynucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, 3'-OMe nucleotides, phosphate group modified nucleotides, terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, 2'-amino modified nucleotides, phosphoramidates, non-natural base comprising nucleotides or 5'-phosphate mimic modified nucleotides; Preferably, the 5'-phosphate mimic is selected from 5'-oxymethylphosphonate, 5'-vinylphosphonate, 5' vinylphosphonate-2'-N-acetyl or 5'-malonylphosphonate.

53. The double-stranded oligonucleotide of any one of claims 1-52, wherein, at least one nucleotide of the double stranded oligonucleotide is conjugated to one or more targeting ligands; Preferably, the targeting ligand is conjugated to the sense strand of the oligonucleotide; Preferably, the targeting ligand is conjugated to any one nucleotide of the sense strand of the oligonucleotide; Preferably, the targeting ligand is conjugated to the 5' end of the sense strand of the oligonucleotide; Preferably, the targeting ligand is conjugated to the 3'-end of the sense strand of the oligonucleotide; 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 has the following structure: Preferably, the targeting ligand is conjugated to the sense strand of the oligonucleotide at the 3'-end via a linker, forming a conjugate as follows: X can be selected from S or O; More preferably, the targeting ligand is compound 2, which has the following structure: Alternatively, the compound 2 is conjugated to the 3'-end of the sense strand of the oligonucleotide.

54. The double stranded oligonucleotide of any one of claims 1-53, which inhibits expression of a target gene in a cell, the target gene selected from the group consisting of: LPA, PNPLA3, ASGR1, F7, F12, FXI, APOCIII, APOB, APOL1, TTR, PCSK9, SCAP, KRAS, CD274, PDCD1, C5, C3, CFB, MASP2, ALAS1, MSTN, HAO1, LDHA, ANGPTL3, SERPINA1, HSD17B13, AGT, HAMP, LECT2, EGFR, VEGF, KIF11, AT3, CTNNB1, HMGB1, HIF1A, APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, SCN9A, SCN10A, CACNA1B, ATXNl, SCA1, SCA7, SCA8, MeCP2, PRNP, SODl, INHBE, CIDEB, DMPK, and STAT3.

55. A composition comprising the double stranded oligonucleotide of any one of claims 1-54, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

56. The composition of claim 55, further comprising one or more additional therapeutic components.

57. The composition of claim 55 or 56, wherein, The composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial.

58. The composition of claim 55 or 56, wherein, The composition is formulated for ocular, vaginal, rectal, intranasal, transdermal, subcutaneous administration, intravenous drip, intra-arterial, perilymphatic, intrabronchial, intrapleural, intraperitoneal, intracerebrospinal, or intramuscular injection, pulmonary, intrathecal, or intraventricular administration.

59. A cell comprising the double stranded oligonucleotide of any one of claims 1-54.

60. A method of inhibiting expression of a target gene in a cell, the method comprising the steps of: delivering to the subject or object the double stranded oligonucleotide of any one of claims 1-54, or the composition of any one of claims 55-58, to inhibit expression of a particular target gene in the subject.

61. The method of claim 60, wherein, The target gene is selected from the group consisting of LPA, PNPLA3, ASGR1, F7, F12, FXI, APOCIII, APOB, APOL1, TTR, PCSK9, SCAP, KRAS, CD274, PDCD1, C5, C3, CFB, MASP2, ALAS1, MSTN, HAO1, LDHA, ANGPTL3, SERPINA1, HSD17B13, AGT, HAMP, LECT2, EGFR, VEGF, KIF11, AT3, CTNNB1, HMGB1, HIF1A, APP, ATXN2, C9orf72, TARDBP, MAPT (Tau), HTT, SNCA, FUS, ATXN3, SCN9A, SCN10A, CACNA1B, ATXNl, SCA1, SCA7, SCA8, MeCP2, PRNP, SODl, INHBE, CIDEB, DMPK, and STAT3.

62. Use of the double-stranded oligonucleotide of any one of claims 1-54 or the composition of any one of claims 55-58 in the manufacture of a medicament for a viral disease, a neuromuscular disease, a bacterial infection, an inflammatory and immune disease, a metabolic disease, a liver disease, a kidney disease, a cardiovascular disease, an ophthalmic disease, a pulmonary disease, a tumor, and a rare disease.

63. A method of treating or preventing a disease or condition in a subject, wherein, The method comprises administering to the subject an amount of the oligonucleotide of any one of claims 1-54 or the composition of any one of claims 55-58 sufficient to inhibit expression of a gene that causes a disease in the subject.

64. The method of claim 63, wherein, The disease or condition is selected from a viral disease, a neuromuscular disease, a bacterial infection, an inflammatory and immune disease, a metabolic disease, a liver disease, a kidney disease, a cardiovascular disease, an ophthalmic disease, a pulmonary disease, a tumor, or a rare disease.

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