Nucleic acids, drug compositions and complexes and methods of preparation and use

Abstract

The present invention provides an siRNA that suppresses the expression of the xanthine oxidase (XO) gene, a drug composition and an siRNA complex, and their use in the preparation of a drug for the treatment and / or prevention of a uric acid metabolism disorder or a disease or physiological condition caused by a uric acid metabolism disorder, wherein each nucleotide of the siRNA is independently modified or unmodified, and the siRNA comprises a sense strand and an antisense strand.

Description

[Technical field]

[0001] The present disclosure relates to nucleic acids capable of suppressing xanthine oxidase (XO) gene expression, pharmaceutical compositions containing the nucleic acids, and siRNA complexes. The present disclosure further relates to methods for preparing and using these nucleic acids, pharmaceutical compositions, and siRNA complexes. [Background technology]

[0002] Gout is a disease that directly correlates with hyperuricemia due to abnormal purine metabolism and / or decreased excretion of uric acid. Gout has long been a common disease in developed countries such as Europe and the United States, and since World War II, its prevalence has been increasing year by year around the world with the economic development of each country, and the age of the patients is also becoming younger. Currently, there are 12 million gout patients in China.

[0003] Xanthine oxidase (XO) is one of the key target points for treating gout. By inhibiting the expression of XO, the production of hypoxanthine and guanine can be effectively inhibited, and the production of uric acid can be reduced, so as to achieve the purpose of alleviating the progression of gout and improving the pathology. By inhibiting the expression of XO gene, diseases caused by abnormal uric acid metabolism at the cellular level, especially hyperuricemia and gout, can be prevented and treated. Small interfering RNA (siRNA) can be used based on the mechanism of RNA interference (RNAi) to sequence-specifically inhibit or block the expression of any target gene of interest, thereby achieving the purpose of treating the disease.

[0004] In the development of siRNA drugs that suppress XO gene expression and treat diseases caused by uric acid metabolic disorders, appropriate siRNA and its modification as well as an effective delivery system are key technologies. Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors of the present disclosure unexpectedly discovered that by having the following siRNA and its modified sequence provided by the present disclosure, the expression of the XO gene can be specifically suppressed, and the drug composition or siRNA complex can specifically target the liver, suppress the expression of the XO gene in the liver, and realize the treatment or prevention of diseases caused by uric acid metabolism disorders, and thus completed the present invention. [Means for solving the problem]

[0006] In some embodiments, the present disclosure provides an siRNA capable of suppressing XO gene expression, the siRNA comprising a sense strand and an antisense strand, each nucleotide of the siRNA being independently modified or unmodified, the sense strand comprising nucleotide sequence I, the antisense strand comprising nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II forming a double-stranded region in a reverse complementary manner at least in part, and the nucleotide sequence I and the nucleotide sequence II being one selected from the sequences shown in i) to xii) below.

[0007] i) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 are equal in length and have no more than three nucleotide differences; 5'-GAGAUGAAGUUCAAGAAUZ 1 -3' (SEQ ID NO: 1), 5'-Z 2 AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 2) However, Z 1 is A and Z 2 is U, and in said nucleotide sequence I, position Z 1 Nucleotide Z corresponding to 3 and in said nucleotide sequence II, position Z 2 Nucleotide Z corresponding to 4 wherein Z 4is the first nucleotide at the 5' end of the antisense strand.

[0008] ii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and have no more than three nucleotide differences; 5'-CAUAACUGGAAUUUGUAAZ 5 -3' (SEQ ID NO: 61), 5'-Z 6 UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:62) However, Z 5 is U and Z 6 is A, and in said nucleotide sequence I, position Z 5 Nucleotide Z corresponding to 7 and in said nucleotide sequence II, position Z 6 Nucleotide Z corresponding to 8 wherein Z 8 is the first nucleotide at the 5' end of the antisense strand.

[0009] iii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 are equal in length and have no more than three nucleotide differences; 5'-CAUUAUCACAAUUGAGGAZ 9 -3' (SEQ ID NO: 121), 5'-Z 10 UCCUCAAUUGUGAUAAUG-3' (SEQ ID NO: 122) However, Z 9 is U and Z 10 is A, and in said nucleotide sequence I, position Z 9 Nucleotide Z corresponding to 11 and in said nucleotide sequence II, position Z 10 Nucleotide Z corresponding to 12 wherein Z12 is the first nucleotide at the 5' end of the antisense strand.

[0010] iv) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 are equal in length and have no more than three nucleotide differences; 5'-GGAUCUCUCUCAGAGUAUZ 13 -3' (SEQ ID NO: 181), 5'-Z 14 AUACUCUGAGAGAGAUCC-3' (SEQ ID NO: 182) However, Z 13 is U and Z 14 is A, and in said nucleotide sequence I, position Z 13 Nucleotide Z corresponding to 15 and in said nucleotide sequence II, position Z 14 Nucleotide Z corresponding to 16 wherein Z 16 is the first nucleotide at the 5' end of the antisense strand.

[0011] v) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 are equal in length and have no more than three nucleotide differences; 5'-ACAUGGACAACUGCUAUAZ 17 -3' (SEQ ID NO: 241), 5'-Z 18 UAUAGCAGUUGUCCAUGU-3' (SEQ ID NO: 242) However, Z 17 is A and Z 18 is U, and in said nucleotide sequence I, position Z 17 Nucleotide Z corresponding to 19 and in said nucleotide sequence II, position Z 18 Nucleotide Z corresponding to 20wherein Z 20 is the first nucleotide at the 5' end of the antisense strand.

[0012] vi) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 302 are equal in length and have no more than three nucleotide differences; 5'-UAGCAAGCUCUCAGUAUCZ 21 -3' (SEQ ID NO: 301), 5'-Z 22 GAUACUGAGAGCUUGCUA-3' (SEQ ID NO: 302) However, Z 21 is A and Z 22 is U, and in said nucleotide sequence I, position Z 21 Nucleotide Z corresponding to 23 and in said nucleotide sequence II, position Z 22 Nucleotide Z corresponding to 24 wherein Z 24 is the first nucleotide at the 5' end of the antisense strand.

[0013] vii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 362 are equal in length and have no more than three nucleotide differences; 5'-AUAAGGUUACUUGUGUUGZ 25 -3' (SEQ ID NO: 361), 5'-Z 26 CAACACAAGUAACCUUAU-3' (SEQ ID NO: 362) However, Z 25 is A and Z 26 is U, and in said nucleotide sequence I, position Z 25 Nucleotide Z corresponding to 27 and in said nucleotide sequence II, position Z 26Nucleotide Z corresponding to 28 wherein Z 28 is the first nucleotide at the 5' end of the antisense strand.

[0014] viii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 422 are equal in length and have no more than three nucleotide differences; 5'-GAAAAUCACCUAUGAAGAZ 29 -3' (SEQ ID NO: 421), 5'-Z 30 UCUUCAUAGGUGAUUUUC-3' (SEQ ID NO: 422) However, Z 29 is A and Z 30 is U, and in said nucleotide sequence I, position Z 29 Nucleotide Z corresponding to 31 and in said nucleotide sequence II, position Z 30 Nucleotide Z corresponding to 32 wherein Z 32 is the first nucleotide at the 5' end of the antisense strand.

[0015] ix) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 481 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 482 are equal in length and have no more than three nucleotide differences; 5'-GAUGCUAUAAAGAACAACZ 33 -3' (SEQ ID NO: 481), 5'-Z 34 GUUGUUCUUUAUAGCAUC-3' (SEQ ID NO: 482) However, Z 33 is U and Z 34 is A, and in said nucleotide sequence I, position Z 33 Nucleotide Z corresponding to 35and in said nucleotide sequence II, position Z 34 Nucleotide Z corresponding to 36 wherein Z 36 is the first nucleotide at the 5' end of the antisense strand.

[0016] x) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 541 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 542 are equal in length and have no more than three nucleotide differences; 5'-GAACAACUCCUUUUAUGGZ 37 -3' (SEQ ID NO: 541), 5'-Z 38 CCAUAAAAGGAGUUGUUC-3' (SEQ ID NO:542) However, Z 37 is A and Z 38 is U, and in said nucleotide sequence I, position Z 37 Nucleotide Z corresponding to 39 and in said nucleotide sequence II, position Z 38 Nucleotide Z corresponding to 40 wherein Z 40 is the first nucleotide at the 5' end of the antisense strand.

[0017] xi) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 601 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 602 are equal in length and have no more than three nucleotide differences; 5'-CUUGCUCUGAAGUAGAAAZ 41 -3' (SEQ ID NO: 601), 5'-Z 42 AUUUCUACUUCAGAGCAAG-3' (SEQ ID NO: 602) However, Z 41 is U and Z 42 is A, and in said nucleotide sequence I, position Z 41 Nucleotide Z corresponding to43 and in said nucleotide sequence II, position Z 42 Nucleotide Z corresponding to 44 wherein Z 44 is the first nucleotide at the 5' end of the antisense strand.

[0018] xii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 661 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 662 are equal in length and have no more than three nucleotide differences; 5'-CUUCUUUGCCAUCAAAGAZ 45 -3' (SEQ ID NO: 661), 5'-Z 46 UCUUUGAUGGCAAAGAAG-3' (SEQ ID NO:662) However, Z 45 is U and Z 46 is A, and in said nucleotide sequence I, position Z 45 Nucleotide Z corresponding to 47 and in said nucleotide sequence II, position Z 46 Nucleotide Z corresponding to 48 wherein Z 48 is the first nucleotide at the 5' end of the antisense strand.

[0019] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a siRNA of the present disclosure and a pharma- ceutically acceptable carrier.

[0020] In some embodiments, the present disclosure provides a siRNA complex comprising an siRNA provided by the present disclosure and a conjugation group conjugated and bound to the siRNA.

[0021] In some embodiments, the present disclosure provides use of the siRNA, and / or drug composition and / or siRNA complex of the present disclosure in the preparation of a drug for the treatment and / or prevention of uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders.

[0022] In some embodiments, the present disclosure provides a method for treating and / or preventing uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders, comprising administering an effective amount of an siRNA, and / or drug composition and / or siRNA complex of the present disclosure to a subject in need thereof.

[0023] In some embodiments, the present disclosure provides a method for inhibiting expression of the XO gene in a hepatocyte, comprising contacting the hepatocyte with an effective amount of an siRNA, and / or drug composition and / or siRNA complex of the present disclosure.

[0024] In some embodiments, the present disclosure provides kits comprising the siRNAs, and / or drug compositions and / or siRNA complexes of the present disclosure. Incorporation by reference

[0025] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Effect of the Invention

[0026] The siRNA, drug composition and siRNA complex provided by the present disclosure have excellent stability, high XO mRNA suppression activity, reduced off-target effects, and / or can significantly treat or alleviate uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders, in particular hyperuricemia and / or gout symptoms.

[0027] In some embodiments, the siRNA, drug composition or siRNA complex provided by the present disclosure exhibits excellent target gene suppression activity in in vitro cell experiments. In some embodiments, the siRNA, drug composition or siRNA complex provided by the present disclosure exhibits a target gene expression suppression rate of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in liver cells. In some embodiments, the siRNA provided by the present disclosure exhibits high suppression activity against XO mRNA in an in vitro psiCHECK system, and exhibits a certain suppression effect against any of the XO target sequences at different siRNA concentrations, particularly at a concentration of 0.1 nM, the suppression rate against the target sequence is at least 61.39%, even as high as 85.43%. In some embodiments, the siRNA provided by the present disclosure exhibits high suppression activity in CAL-27 cells, and exhibits an IC 50 is 0.037-0.3277 μM. In some embodiments, the siRNA complex provided by the present disclosure exhibits high inhibitory activity in mouse primary hepatocytes, and the inhibition rate against XO mRNA is at least 78.95%, even as high as 88.07%, at an siRNA concentration of 20 nM.

[0028] In some embodiments, the siRNA, drug composition or siRNA complex provided by the present disclosure can have higher stability and / or higher activity in vivo. In some embodiments, the siRNA, drug composition or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% target gene expression suppression rate in vivo. In some embodiments, the siRNA, drug composition or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% XO gene expression suppression rate in vivo. In some embodiments, the siRNA, drug composition or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% intrahepatic XO gene expression suppression rate in vivo. In some embodiments, the siRNA, drug composition, or siRNA complex provided by the present disclosure exhibits an intrahepatic XO gene expression suppression rate in an animal model of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, drug composition, or siRNA complex provided by the present disclosure exhibits an intrahepatic XO gene expression suppression rate in a human subject of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA complex provided by the present disclosure exhibits an in vivo XO mRNA expression suppression rate of 70.9-76.2% in mice at an siRNA concentration of 3 mg / kg.

[0029] In some embodiments, the siRNA, drug composition or siRNA complex provided by the present disclosure does not show significant off-target effect. Off-target effect can be, for example, the inhibition of the normal expression of non-target genes. When the binding / inhibition of off-target gene expression is 50%, 40%, 30%, 20% or 10% lower than the on-target gene effect, the off-target effect is considered to be not significant.

[0030] Thus, the siRNA, pharmaceutical composition and siRNA complex provided by the present disclosure can suppress the expression of the XO gene and effectively treat and / or prevent uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders, and have bright prospects for application.

[0031] Other features and advantages of the present disclosure are described in detail in the Detailed Description section that follows. [Brief description of the drawings]

[0032] [Fig. 1A-1F] Dose-effect curves fitted according to the relative expression levels of XO mRNA in CAL-27 cells in vitro after transfection with different siRNAs. [Diagram 2] 1 is a histogram of relative expression levels of XO mRNA in mouse primary hepatocytes after transfection with different siRNAs. [Diagram 3] FIG. 13 is a scatter plot of relative expression levels of XO mRNA in mice after administration of 3 mg / kg of different siRNA complexes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The following describes in detail the embodiments of the present disclosure. It should be understood that the embodiments of the present disclosure are merely for explaining or interpreting the present disclosure and are not intended to limit the present disclosure.

[0034] As used herein, XO mRNA refers to the mRNA having the sequence set forth in Genbank Accession No. NM_000379.3. Furthermore, unless otherwise specified, the term "target gene" as used in this disclosure refers to a gene that transcribes the XO mRNA, and the term "target mRNA" refers to the XO mRNA.

[0035] <Definition> Unless otherwise specified in the context, capital letters C, G, U, and A represent the base sequence of nucleotides, lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, lower case letter s represents that two nucleotides adjacent to the left and right sides of the letter s are bonded by a thiophosphate group, P1 represents that one nucleotide adjacent to the right side of the P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide, the combination letter VP represents that one nucleotide adjacent to the right side of the combination letter VP is a vinyl phosphate-modified nucleotide, the combination letter Ps represents that one nucleotide adjacent to the right side of the combination letter Ps is a thiophosphate-modified nucleotide, and capital letter P represents that one nucleotide adjacent to the right side of the letter P is a 5'-phosphate nucleotide.

[0036] In this context, the "fluoro-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced with fluorine, and the "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced with a non-fluorine group. The "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide, such as an isonucleotide, bridged nucleic acid (BNA) or acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced with methoxy.

[0037] In the context of this specification, the terms "complementary" or "reverse complementary" may be used interchangeably and have the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, each base of one strand is paired with a base on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair contains one purine and one pyrimidine. If adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, it is considered that both strands are complementary and that the sequence of the strand can be deduced from the sequence of the complementary strand. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, bases at corresponding positions are not paired in a complementary manner.

[0038] Unless otherwise specified in context, "essentially reverse complementary" refers to no more than three base mismatches between two related nucleotide sequences, "substantially reverse complementary" refers to no more than one base mismatch between two nucleotide sequences, and "fully reverse complementary" refers to no base mismatches between two nucleotide sequences.

[0039] In this context, the presence of a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter, for example, when one nucleotide base in the latter is A, a nucleotide difference is recognized to exist between two nucleotide sequences at that position if the corresponding nucleotide base at the same position in the former is U, C, G, or T. In some embodiments, a nucleotide difference at a position is also considered to have occurred when an abasic nucleotide or its equivalent is used in place of the nucleotide at the original position.

[0040] In the context, particularly in describing the preparation method of the siRNA, drug composition or siRNA complex of the present disclosure, unless otherwise specified, the nucleoside monomer refers to modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, RNA phosphoramidites are sometimes referred to as nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis according to the type and order of nucleotides in the siRNA or siRNA complex to be prepared. Phosphoramidite solid-phase synthesis is a method used for RNA synthesis known to those skilled in the art. All of the nucleoside monomers used in the present disclosure are commercially available.

[0041] In the context of this specification, unless otherwise specified, "conjugate" refers to two or more chemical moieties each having a specific function being covalently bonded to each other, and accordingly, "complex" refers to a compound formed by covalently bonding the respective chemical moieties. Furthermore, "siRNA complex" refers to a compound formed by covalently bonding one or more chemical moieties having a specific function to siRNA. In the following text, the siRNA complex of the present disclosure may also be simply referred to as "complex". Depending on the context, the siRNA complex is understood to be a general term for siRNA complexes, a general term for siRNA complexes represented by formula (305) and formula (307), or a siRNA complex represented by formula (305), formula (307), or formula (308). In the context of this specification, a "complex molecule" should be understood to be a specific compound that can be reacted to be conjugated to siRNA to finally form the siRNA complex of the present disclosure.

[0042] As used herein, "optional" or "optionally" refers to the subsequently described event or circumstance that may or may not occur, and includes the occurrence and non-occurrence of the described event or circumstance. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined in the following sentence. With respect to any groups that contain one or more substituents, it will be understood by those of skill in the art that it is not intended that these groups introduce any substitutions or substitution patterns that are sterically impractical, synthetically impractical, and / or inherently unstable.

[0043] As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, 1 to 8, or 1 to 6 carbon atoms. For example, C 1 -C 6 Alkyl includes straight and branched chain alkyls of 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to include all branched and straight chain forms having that number of carbons. Thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to a residue similar to alkyl but having two points of attachment.

[0044] As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl having at least one carbon-carbon double bond, which is obtained by removing one hydrogen molecule from adjacent carbon atoms of a parent alkyl. The group may be in the cis or trans configuration of the double bond. Exemplary alkenyls include, but are not limited to, vinyl, propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, and butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, and buta-1,3-dien-2-yl. In some embodiments, alkenyl has from 2 to 20 carbon atoms, but in other embodiments, from 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl, referring to residues similar to alkenyl, but having two points of attachment.

[0045] As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond, the carbon-carbon triple bond being obtained by removing two hydrogen molecules from adjacent carbon atoms of a parent alkyl. Exemplary alkynyls include, but are not limited to, ethynyl, propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl, and butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl. In certain embodiments, alkynyl has 2-20 carbon atoms, while in other embodiments, 2-10, 2-8, or 2-6 carbon atoms. Alkynylene is a subset of alkynyl and refers to a residue similar to alkynyl but having two points of attachment.

[0046] As used herein, "alkoxy" refers to an alkyl of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, etc. An alkoxy typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.

[0047] As used herein, "aryl" refers to a group formed by removing a hydrogen atom from a ring carbon atom derived from an aromatic monocyclic or polycyclic hydrocarbon ring system. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon from 6 to 18 carbon atoms, and at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Aryl includes, but is not limited to, groups such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to a residue that is the same as aryl but has two points of attachment.

[0048] As used herein, a "halogen substituent" or "halo" refers to fluoro, chloro, bromo, or iodo, and the term "halogen" includes fluorine, chlorine, bromine, or iodine.

[0049] As used herein, "halogenated alkyl" refers to an alkyl group as defined above in which a specified number of carbon atoms are substituted with one or more halogen atoms, up to the maximum permitted number. Illustrative examples of halogenated alkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, or pentafluoroethyl.

[0050] "Heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, or sulfur. Unless otherwise stated in the specification, a heterocyclic group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, and may include fused or bridged ring systems. The heteroatoms in a heterocyclic group may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. A heterocyclic group is partially saturated or fully saturated. A heterocyclic group may be attached to the remainder of the molecule through any atom of the ring. Illustrative examples of such heterocyclic groups include dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindole, octahydroisoindole, 2-oxapiperazinyl, 2-oxapiperidyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, and the like. These include, but are not limited to, lysinyl, pyrazolidyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl.

[0051] "Heteroaryl" refers to a group derived from a 3-18 membered aromatic ring radical containing 2-17 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system in which at least one of the rings is fully unsaturated, i.e., it contains a cyclic delocalized (4n+2) π-electron system according to the Huckel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heteroaryl is bonded to the remainder of the molecule through any atom in the ring. Illustrative examples of heteroaryl include azepinyl, acridinyl, benzimidazolyl, benzoindole, 1,3-benzodioxazolyl, benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxy, and the like. benzodioxinyl, benzopyranyl, benzopyronyl, benzofuryl, benzofuranonyl, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl(5,6 dihydrobenzo[h]cinnolinyl), 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuryl, dibenzothiophenyl, furyl, furanonyl, furo[3,2-c]pyridyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridyl, isothiazolyl, imidazolyl, indazolyl, indole, isoindole, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl linyl), naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, Pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolyl, tetrahydroquinolyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7 ,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl / thienyl.

[0052] A variety of hydroxy protecting groups can be used in the present disclosure. Generally, a protecting group can render a chemical functionality insensitive to a particular reaction condition, and can be added to and removed from that functionality in a molecule without substantially damaging the remainder of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), or 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), or TMTr (4,4',4''-trimethoxytrityl).

[0053] The term "subject" as used herein refers to any animal, e.g., a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, or any type of poultry.

[0054] As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including, but not limited to, a therapeutic effect. A "therapeutic effect" refers to eradicating or ameliorating the underlying disorder being treated. A therapeutic effect is also obtained by observing an improvement in a subject by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, even though the subject may still be afflicted by the underlying disorder.

[0055] As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a prophylactic effect. To obtain a "prophylactic effect," siRNA, siRNA complexes, or drug compositions can be administered to a subject at risk of suffering from a particular disease, although the subject may not have been diagnosed with the disease, or to a subject who has been reported to have one or more pathological symptoms of the disease.

[0056] In one aspect, the present disclosure provides siRNA Nos. 1 to 12 capable of suppressing XO gene expression, which will be described in detail below in order.

[0057] The siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group, and a base, which is known to those skilled in the art, and therefore will not be described here.

[0058] <First siRNA> According to the present disclosure, the siRNA may be a first siRNA.

[0059] The first siRNA comprises a sense strand and an antisense strand, each nucleotide of the first siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 are equal in length and have no more than three nucleotide differences. 5'-GAGAUGAAGUUCAAGAAUZ 1 -3' (SEQ ID NO: 1), 5'-Z 2 AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 2) However, Z 1 is A and Z 2 is U, and in said nucleotide sequence I, position Z 1 Nucleotide Z corresponding to 3 and in said nucleotide sequence II, position Z 2 Nucleotide Z corresponding to 4 wherein Z 4 is the first nucleotide at the 5' end of the antisense strand.

[0060] In this context, "corresponding in position" refers to being at the same position in a nucleotide sequence from the same end of the nucleotide sequence. For example, the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide that corresponds in position to the first nucleotide at the 3' end of SEQ ID NO:1.

[0061] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0062] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:1, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:2.

[0063] In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:2 is Z 4 including the difference at the position of Z 4 is selected from A, C, or G. In some embodiments, the nucleotide difference is 4 is the difference in position, Z 4 is selected from A, C, or G. In some embodiments, Z 3 is Z 4 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0064] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are basically reverse complementary, substantially reverse complementary, or completely reverse complementary, where basically reverse complementary means that there are no more than three base mismatches between the two nucleotide sequences, substantially reverse complementary means that there are no more than one base mismatch between the two nucleotide sequences, and completely reverse complementary means that there are no base mismatches between the two nucleotide sequences.

[0065] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:3 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:4. 5'-GAGAUGAAGUUCAAGAAUZ 3 -3' (SEQ ID NO: 3), 5'-Z 4 AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 4) However, the above Z 4is the first nucleotide at the 5' end of the antisense strand, and Z 4 is selected from A, U, G or C; Z 3 is Z 4 and in some embodiments, Z 3 is A and Z 4 is U.

[0066] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure may be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 23, or 23 / 25.

[0067] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV each have a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV and a second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0068] In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is U and the base of the nucleotide sequence IV is A, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is UU and the base sequence of the nucleotide sequence IV is AA from the 5' end to the 3' end, and the length ratio of the sense strand to the antisense strand is 21 / 21; or The nucleotide sequences III and IV are each 3 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is AUU and the base sequence of nucleotide sequence IV is AAU, and the length ratio of the sense strand to the antisense strand is 22 / 22, or the nucleotide sequences III and IV are each 4 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is CAUU and the base sequence of nucleotide sequence IV is AAUG, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and IV are each 2 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is UU and the base sequence of nucleotide sequence IV is AA, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0069] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0070] <Second siRNA> According to the present disclosure, the siRNA may be a second siRNA.

[0071] The second siRNA comprises a sense strand and an antisense strand, each nucleotide of the second siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and have no more than three nucleotide differences. 5'-CAUAACUGGAAUUUGUAAZ 5 -3' (SEQ ID NO: 61), 5'-Z 6 UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:62) However, Z 5 is U and Z 6 is A, and in said nucleotide sequence I, position Z 5 Nucleotide Z corresponding to 7 and in said nucleotide sequence II, position Z 6 Nucleotide Z corresponding to 8 wherein Z 8 is the first nucleotide at the 5' end of the antisense strand.

[0072] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0073] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:61, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:62.

[0074] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 62 is Z8 including the difference at the position of Z 8 is selected from U, C, or G. In some embodiments, the nucleotide difference is 8 is the difference in position, Z 8 is selected from U, C, or G. In some embodiments, Z 7 is Z 8 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0075] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0076] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:63 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:64. 5'-CAUAACUGGAAUUUGUAAZ 7 -3' (SEQ ID NO: 63), 5'-Z 8 UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:64) However, the above Z 8 is the first nucleotide at the 5' end of the antisense strand, and Z 8 is selected from A, U, G or C; Z 7 is Z 8 and in some embodiments, Z 7 is U and Z 8 is A.

[0077] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0078] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV having a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV being equal in length and being substantially reverse complementary or completely reverse complementary, the nucleotide sequence III being bound to the 5' end of the nucleotide sequence I, the nucleotide sequence IV being bound to the 3' end of the nucleotide sequence II, the nucleotide sequence IV being substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence referring to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 61 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0079] In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is A and the base of the nucleotide sequence IV is U, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is AA from the 5' end to the 3' end, and the base sequence of the nucleotide sequence IV is UU, and the length ratio of the sense strand to the antisense strand is 21 / 21; or The nucleotide sequences III and IV are each 3 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is UAA and the base sequence of nucleotide sequence IV is UUA, and the length ratio of the sense strand to the antisense strand is 22 / 22, or the nucleotide sequences III and IV are each 4 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is GUAA and the base sequence of nucleotide sequence IV is UUAC, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and IV are each 2 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is AA and the base sequence of nucleotide sequence IV is UU, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0080] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0081] <Third siRNA> According to the present disclosure, the siRNA may be a third siRNA.

[0082] The third siRNA comprises a sense strand and an antisense strand, each nucleotide of the third siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 are equal in length and have no more than three nucleotide differences. 5'-CAUUAUCACAAUUGAGGAZ 9 -3' (SEQ ID NO: 121), 5'-Z 10 UCCUCAAUUGUGAUAAUG-3' (SEQ ID NO: 122) However, Z 9 is U and Z 10 is A, and in said nucleotide sequence I, position Z 9 Nucleotide Z corresponding to 11 and in said nucleotide sequence II, position Z 10 Nucleotide Z corresponding to 12 wherein Z 12 is the first nucleotide at the 5' end of the antisense strand.

[0083] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0084] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:121, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:122.

[0085] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 122 is Z12 including the difference at the position of Z 12 is selected from U, C, or G. In some embodiments, the nucleotide difference is 12 is the difference in position, Z 12 is selected from U, C, or G. In some embodiments, Z 11 is Z 12 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0086] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0087] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:123 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:124. 5'-CAUUAUCACAAUUGAGGAZ 11 -3' (SEQ ID NO: 123), 5'-Z 12 UCCUCAAUUGUGAUAAUG-3' (SEQ ID NO: 124) However, the above Z 12 is the first nucleotide at the 5' end of the antisense strand, and Z 12 is selected from A, U, G or C; Z 11 is Z 12 and in some embodiments, Z 11 is U and Z 12 is A.

[0088] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0089] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II, the nucleotide sequence IV and a second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 121 in the target mRNA and has a length equal to that of the nucleotide sequence IV.

[0090] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is C, and the base of the nucleotide sequence IV is G, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is GC from the 5' end to the 3' end, and the base sequence of the nucleotide sequence IV is GC, and the length ratio of the sense strand to the antisense strand is 21 / 21. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is AGC and the base sequence of nucleotide sequence IV is GCU, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is CAGC and the base sequence of nucleotide sequence IV is GCUG, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, nucleotide sequences III and IV are each 2 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is GC and the base sequence of nucleotide sequence IV is GC, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0091] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0092] <The fourth siRNA> According to the present disclosure, the siRNA may be a fourth siRNA.

[0093] The fourth siRNA comprises a sense strand and an antisense strand, each nucleotide of the fourth siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 are equal in length and have no more than three nucleotide differences. 5'-GGAUCUCUCUCAGAGUAUZ 13 -3' (SEQ ID NO: 181), 5'-Z 14 AUACUCUGAGAGAGAUCC-3' (SEQ ID NO: 182) However, Z 13 is U and Z 14 is A, and in said nucleotide sequence I, position Z 13 Nucleotide Z corresponding to 15 and in said nucleotide sequence II, position Z 14 Nucleotide Z corresponding to 16 wherein Z 16 is the first nucleotide at the 5' end of the antisense strand.

[0094] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0095] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:181, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:182.

[0096] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 182 is Z16 including the difference at the position of Z 16 is selected from U, C, or G. In some embodiments, the nucleotide difference is 16 is the difference in position, Z 16 is selected from U, C, or G. In some embodiments, Z 15 is Z 16 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0097] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0098] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:183 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:184. 5'-GGAUCUCUCUCAGAGUAUZ 15 -3' (SEQ ID NO: 183), 5'-Z 16 AUACUCUGAGAGAGAUCC-3' (SEQ ID NO: 184) However, the above Z 16 is the first nucleotide at the 5' end of the antisense strand, and Z 16 is selected from A, U, G or C; Z 15 is Z 16 and in some embodiments, Z 15 is U and Z 16 is A.

[0099] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0100] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV having a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV being equal in length and being substantially reverse complementary or completely reverse complementary, the nucleotide sequence III being bound to the 5' end of the nucleotide sequence I, the nucleotide sequence IV being bound to the 3' end of the nucleotide sequence II, the nucleotide sequence IV being substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence referring to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 181 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0101] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is A and the base of the nucleotide sequence IV is U, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is CA and the base sequence of the nucleotide sequence IV is UG, and the length ratio of the sense strand to the antisense strand is 21 / 21. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is CCA and the base sequence of nucleotide sequence IV is UGG, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is CCCA and the base sequence of nucleotide sequence IV is UGGG, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, nucleotide sequences III and IV are each 2 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is CA and the base sequence of nucleotide sequence IV is UG, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0102] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0103] <The fifth siRNA> According to the present disclosure, the siRNA may be a fifth siRNA.

[0104] The fifth siRNA comprises a sense strand and an antisense strand, each nucleotide of the fifth siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 are equal in length and have no more than three nucleotide differences. 5'-ACAUGGACAACUGCUAUAZ 17 -3' (SEQ ID NO: 241), 5'-Z 18 UAUAGCAGUUGUCCAUGU-3' (SEQ ID NO: 242) However, Z 17 is A and Z 18 is U, and in said nucleotide sequence I, position Z 17 Nucleotide Z corresponding to 19 and in said nucleotide sequence II, position Z 18 Nucleotide Z corresponding to 20 wherein Z 20 is the first nucleotide at the 5' end of the antisense strand.

[0105] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0106] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:241, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:242.

[0107] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 242 is Z20 including the difference at the position of Z 20 is selected from A, C, or G. In some embodiments, the nucleotide difference is 20 is the difference in position, Z 20 is selected from A, C, or G. In some embodiments, Z 19 is Z 20 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0108] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0109] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:243 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:244. 5'-ACAUGGACAACUGCUAUAZ 19 -3' (SEQ ID NO: 243), 5'-Z 20 UAUAGCAGUUGUCCAUGU-3' (SEQ ID NO: 244) However, the above Z 20 is the first nucleotide at the 5' end of the antisense strand, and Z 20 is selected from A, U, G or C; Z 19 is Z 20 and in some embodiments, Z 19 is A and Z 20 is U.

[0110] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0111] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II, the nucleotide sequence IV and a second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 241 in the target mRNA and has a length equal to that of the nucleotide sequence IV.

[0112] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is C and the base of the nucleotide sequence IV is G, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is CC and the base sequence of the nucleotide sequence IV is GG, and the length ratio of the sense strand to the antisense strand is 21 / 21, in the 5'-3' direction. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, and from the 5' to 3' end, the base sequence of nucleotide sequence III is UCC and the base sequence of nucleotide sequence IV is GGA, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, and from the 5' to 3' end, the base sequence of nucleotide sequence III is UUCC and the base sequence of nucleotide sequence IV is GGAA, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, nucleotide sequences III and IV are each 2 nucleotides in length, and from the 5' to 3' end, the base sequence of nucleotide sequence III is CC and the base sequence of nucleotide sequence IV is GG, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0113] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0114] <6th siRNA> According to the present disclosure, the siRNA may be a sixth siRNA.

[0115] The sixth siRNA comprises a sense strand and an antisense strand, each nucleotide of the sixth siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 302 are equal in length and have no more than three nucleotide differences. 5'-UAGCAAGCUCUCAGUAUCZ 21 -3' (SEQ ID NO: 301), 5'-Z 22 GAUACUGAGAGCUUGCUA-3' (SEQ ID NO: 302) However, Z 21 is A and Z 22 is U, and in said nucleotide sequence I, position Z 21 Nucleotide Z corresponding to 23 and in said nucleotide sequence II, position Z 22 Nucleotide Z corresponding to 24 wherein Z 24 is the first nucleotide at the 5' end of the antisense strand.

[0116] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0117] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:301, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:302.

[0118] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 302 is Z24 including the difference at the position of Z 24 is selected from A, C, or G. In some embodiments, the nucleotide difference is 24 is the difference in position, Z 24 is selected from A, C, or G. In some embodiments, Z 23 is Z 24 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0119] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0120] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:303 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:304. 5'-UAGCAAGCUCUCAGUAUCZ 23 -3' (SEQ ID NO: 303), 5'-Z 24 GAUACUGAGAGCUUGCUA-3' (SEQ ID NO: 304) However, the above Z 24 is the first nucleotide at the 5' end of the antisense strand, and Z 24 is selected from A, U, G or C; Z 23 is Z 24 and in some embodiments, Z 23 is A and Z 24 is U.

[0121] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0122] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each 1 to 4 nucleotides in length, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II, the nucleotide sequence IV and a second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 301 in the target mRNA and has a length equal to that of the nucleotide sequence IV.

[0123] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is C and the base of the nucleotide sequence IV is G, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is CC and the base sequence of the nucleotide sequence IV is GG, and the length ratio of the sense strand to the antisense strand is 21 / 21, in the 5'-3' direction. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is GCC and the base sequence of nucleotide sequence IV is GGC, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is UGCC and the base sequence of nucleotide sequence IV is GGCA, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, nucleotide sequences III and IV are each 2 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is CC and the base sequence of nucleotide sequence IV is GG, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0124] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0125] <7th siRNA> According to the present disclosure, the siRNA may be a seventh siRNA.

[0126] The seventh siRNA comprises a sense strand and an antisense strand, each nucleotide of the seventh siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 362 are equal in length and have no more than three nucleotide differences. 5'-AUAAGGUUACUUGUGUUGZ 25 -3' (SEQ ID NO: 361), 5'-Z 26 CAACACAAGUAACCUUAU-3' (SEQ ID NO: 362) However, Z 25 is G and Z 26 is C, and in said nucleotide sequence I, position Z 25 Nucleotide Z corresponding to 27 and in said nucleotide sequence II, position Z 26 Nucleotide Z corresponding to 28 wherein Z 28 is the first nucleotide at the 5' end of the antisense strand.

[0127] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0128] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:361, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:362.

[0129] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 362 is Z28 including the difference at the position of Z 28 is selected from A, U, or G. In some embodiments, the nucleotide difference is 28 is the difference in position, Z 28 is selected from A, U, or G. In some embodiments, Z 27 is Z 28 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0130] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0131] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:363 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:364. 5'-AUAAGGUUACUUGUGUUGZ 27 -3' (SEQ ID NO: 363), 5'-Z 28 CAACACAAGUAACCUUAU-3' (SEQ ID NO: 364) However, the above Z 28 is the first nucleotide at the 5' end of the antisense strand, and Z 28 is selected from A, U, G or C; Z 27 is Z 28 and in some embodiments, Z 27 is G and Z 28 is C.

[0132] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0133] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV each have a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 361 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0134] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is G and the base of the nucleotide sequence IV is C, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is GG and the base sequence of the nucleotide sequence IV is CC, and the length ratio of the sense strand to the antisense strand is 21 / 21, in the 5'-3' direction. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, and from the 5' to 3' end, the base sequence of nucleotide sequence III is AGG and the base sequence of nucleotide sequence IV is CCU, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, and from the 5' to 3' end, the base sequence of nucleotide sequence III is AAGG and the base sequence of nucleotide sequence IV is CCUU, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and IV are each 2 nucleotides in length, and from the 5' to 3' end, the base sequence of nucleotide sequence III is GG and the base sequence of nucleotide sequence IV is CC, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0135] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0136] <8th siRNA> According to the present disclosure, the siRNA may be an eighth siRNA.

[0137] The eighth siRNA comprises a sense strand and an antisense strand, each nucleotide of the eighth siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 422 are equal in length and have no more than three nucleotide differences. 5'-GAAAAUCACCUAUGAAGAZ 29 -3' (SEQ ID NO: 421), 5'-Z 30 UCUUCAUAGGUGAUUUUC-3' (SEQ ID NO: 422) However, Z 29 is A and Z 30 is U, and in said nucleotide sequence I, position Z 29 Nucleotide Z corresponding to 31 and in said nucleotide sequence II, position Z 30 Nucleotide Z corresponding to 32 wherein Z 32 is the first nucleotide at the 5' end of the antisense strand.

[0138] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0139] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:421, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:422.

[0140] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 422 is Z32 including the difference at the position of Z 32 is selected from A, C, or G. In some embodiments, the nucleotide difference is 32 is the difference in position, Z 32 is selected from A, C, or G. In some embodiments, Z 31 is Z 32 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0141] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0142] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:423 and said nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:424. 5'-GAAAAUCACCUAUGAAGAZ 31 -3' (SEQ ID NO: 423), 5'-Z 32 UCUUCAUAGGUGAUUUUC-3' (SEQ ID NO: 424) However, the above Z 32 is the first nucleotide at the 5' end of the antisense strand, and Z 32 is selected from A, U, G or C; Z 31 is Z 32 and in some embodiments, Z 31 is A and Z 32 is U.

[0143] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0144] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV each have a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 421 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0145] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is U and the base of the nucleotide sequence IV is A, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is GU and the base sequence of the nucleotide sequence IV is AC, in the 5'-3' direction, the length ratio of the sense strand to the antisense strand is 21 / 21. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is GGU and the base sequence of nucleotide sequence IV is ACC, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is GGGU and the base sequence of nucleotide sequence IV is ACCC, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and IV are each 2 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is GU and the base sequence of nucleotide sequence IV is AC, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0146] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0147] <9th siRNA> According to the present disclosure, the siRNA may be a ninth siRNA.

[0148] The ninth siRNA comprises a sense strand and an antisense strand, each nucleotide of the ninth siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 481 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 482 are equal in length and have no more than three nucleotide differences. 5'-GAUGCUAUAAAGAACAACZ 33 -3' (SEQ ID NO: 481), 5'-Z 34 GUUGUUCUUUAUAGCAUC-3' (SEQ ID NO: 482) However, Z 33 is U and Z 34 is A, and in said nucleotide sequence I, position Z 33 Nucleotide Z corresponding to 35 and in said nucleotide sequence II, position Z 34 Nucleotide Z corresponding to 36 wherein Z 36 is the first nucleotide at the 5' end of the antisense strand.

[0149] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0150] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:481, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:482.

[0151] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 482 is Z36 including the difference at the position of Z 36 is selected from U, C, or G. In some embodiments, the nucleotide difference is 36 is the difference in position, Z 36 is selected from U, C, or G. In some embodiments, Z 35 is Z 36 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0152] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0153] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:483 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:484. 5'-GAUGCUAUAAAGAACAACZ 35 -3' (SEQ ID NO: 483), 5'-Z 36 GUUGUUCUUUAUAGCAUC-3' (SEQ ID NO: 484) However, the above Z 36 is the first nucleotide at the 5' end of the antisense strand, and Z 36 is selected from A, U, G or C; Z 35 is Z 36 and in some embodiments, Z 35 is U and Z 36 is A.

[0154] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0155] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV each have a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 481 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0156] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is G and the base of the nucleotide sequence IV is C, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is AG and the base sequence of the nucleotide sequence IV is CU, and the length ratio of the sense strand to the antisense strand is 21 / 21, in the 5'-3' direction. or nucleotide sequences III and IV are each 3 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is GAG and the base sequence of nucleotide sequence IV is CUC, and the length ratio of the sense strand to the antisense strand is 22 / 22; or nucleotide sequences III and IV are each 4 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is UGAG and the base sequence of nucleotide sequence IV is CUCA, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and IV are each 2 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is AG and the base sequence of nucleotide sequence IV is CU, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0157] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0158] <10th siRNA> According to the present disclosure, the siRNA may be a tenth siRNA.

[0159] The tenth siRNA comprises a sense strand and an antisense strand, each nucleotide of the tenth siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 541 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 542 are equal in length and have no more than three nucleotide differences. 5'-GAACAACUCCUUUUAUGGZ 37 -3' (SEQ ID NO: 541), 5'-Z 38 CCAUAAAAGGAGUUGUUC-3' (SEQ ID NO:542) However, Z 37 is A and Z 38 is U, and in said nucleotide sequence I, position Z 37 Nucleotide Z corresponding to 39 and in said nucleotide sequence II, position Z 38 Nucleotide Z corresponding to 40 wherein Z 40 is the first nucleotide at the 5' end of the antisense strand.

[0160] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0161] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:541, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:542.

[0162] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 542 is Z 40 including the difference at the position of Z 40 is selected from A, C, or G. In some embodiments, the nucleotide difference is 40 is the difference in position, Z 40 is selected from A, C, or G. In some embodiments, Z 39 is Z 40 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0163] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0164] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:543 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:544. 5'-GAACAACUCCUUUUAUGGZ 39 -3' (SEQ ID NO: 543), 5'-Z 40 CCAUAAAAGGAGUUGUUC-3' (SEQ ID NO:544) However, the above Z 40 is the first nucleotide at the 5' end of the antisense strand, and Z 40 is selected from A, U, G or C; Z 39 is Z 40 and in some embodiments, Z 39 is A and Z 40 is U.

[0165] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0166] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV each have a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 541 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0167] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is A and the base of the nucleotide sequence IV is U, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is AA and the base sequence of the nucleotide sequence IV is UU, and the length ratio of the sense strand to the antisense strand is 21 / 21, in the 5'-3' direction. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is AAA and the base sequence of nucleotide sequence IV is UUU, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is UAAA and the base sequence of nucleotide sequence IV is UUUA, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, nucleotide sequences III and IV are each 2 nucleotides in length, from the 5'-end to the 3'-end, the base sequence of nucleotide sequence III is AA and the base sequence of nucleotide sequence IV is UU, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0168] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0169] <11th siRNA> According to the present disclosure, the siRNA may be an eleventh siRNA.

[0170] The 11th siRNA comprises a sense strand and an antisense strand, each nucleotide of the 11th siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 601 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 602 are equal in length and have no more than three nucleotide differences. 5'-CUUGCUCUGAAGUAGAAAZ 41 -3' (SEQ ID NO: 601), 5'-Z 42 AUUUCUACUUCAGAGCAAG-3' (SEQ ID NO: 602) However, Z 41 is U and Z 42 is A, and in said nucleotide sequence I, position Z 41 Nucleotide Z corresponding to 43 and in said nucleotide sequence II, position Z 42 Nucleotide Z corresponding to 44 wherein Z 44 is the first nucleotide at the 5' end of the antisense strand.

[0171] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0172] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:601, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:602.

[0173] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 602 is Z 44 including the difference at the position of Z 44 is selected from U, C, or G. In some embodiments, the nucleotide difference is 44 is the difference in position, Z 44 is selected from U, C, or G. In some embodiments, Z 43 is Z 44 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0174] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0175] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:603 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:604. 5'-CUUGCUCUGAAGUAGAAAZ 43 -3' (SEQ ID NO: 603), 5'-Z 44 UUUCUACUUCAGAGCAAG-3' (SEQ ID NO:604) However, the above Z 44 is the first nucleotide at the 5' end of the antisense strand, and Z 44 is selected from A, U, G or C; Z 43 is Z 44 and in some embodiments, Z 43 is U and Z 44 is A.

[0176] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0177] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV each have a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 601 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0178] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is G and the base of the nucleotide sequence IV is C, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is GG and the base sequence of the nucleotide sequence IV is CC, and the length ratio of the sense strand to the antisense strand is 21 / 21, in the 5'-3' direction. Alternatively, nucleotide sequences III and IV are each 3 nucleotides in length, and from the 5' to the 3' end, the base sequence of nucleotide sequence III is UGG and the base sequence of nucleotide sequence IV is CCA, and the length ratio of the sense strand to the antisense strand is 22 / 22, or nucleotide sequences III and IV are each 4 nucleotides in length, and from the 5' to the 3' end, the base sequence of nucleotide sequence III is GUGG and the base sequence of nucleotide sequence IV is CCAC, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and IV are each 2 nucleotides in length, and from the 5' to the 3' end, the base sequence of nucleotide sequence III is GG and the base sequence of nucleotide sequence IV is CC, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0179] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0180] <12th siRNA> According to the present disclosure, the siRNA may be a twelfth siRNA.

[0181] The 12th siRNA comprises a sense strand and an antisense strand, each nucleotide of the 12th siRNA is independently modified or unmodified, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse complementary manner at least in part, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 661 are equal in length and have no more than three nucleotide differences, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 662 are equal in length and have no more than three nucleotide differences. 5'-CUUCUUUGCCAUCAAAGAZ 45 -3' (SEQ ID NO: 661), 5'-Z 46 UCUUUGAUGGCAAAGAAG-3' (SEQ ID NO:662) However, Z 45 is U and Z 46 is A, and in said nucleotide sequence I, position Z 45 Nucleotide Z corresponding to 47 and in said nucleotide sequence II, position Z 46 Nucleotide Z corresponding to 48 wherein Z 48 is the first nucleotide at the 5' end of the antisense strand.

[0182] In some embodiments, the sense strand comprises only nucleotide sequence I and the antisense strand comprises only nucleotide sequence II.

[0183] In some embodiments, there is no more than one nucleotide difference between nucleotide sequence I and the nucleotide sequence set forth in SEQ ID NO:661, and / or there is no more than one nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:662.

[0184] In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 662 is Z 48 including the difference at the position of Z 48 is selected from U, C, or G. In some embodiments, the nucleotide difference is 48 is the difference in position, Z 48 is selected from U, C, or G. In some embodiments, Z 47 is Z 48 The siRNAs having the above nucleotide differences have high target mRNA suppression ability, and the siRNAs containing these nucleotide differences are also within the scope of protection of the present disclosure.

[0185] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially reverse complementary, or completely reverse complementary.

[0186] In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:663 and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:664. 5'-CUUCUUUGCCAUCAAAGAZ 47 -3' (SEQ ID NO: 663), 5'-Z 48 UCUUUGAUGGCAAAGAAG-3' (SEQ ID NO:664) However, the above Z 48 is the first nucleotide at the 5' end of the antisense strand, and Z 48 is selected from A, U, G or C; Z 47 is Z 48 and in some embodiments, Z 47 is U and Z 48 is A.

[0187] The sense strand and the antisense strand may have the same or different lengths, with the sense strand having a length of 19 to 23 nucleotides and the antisense strand having a length of 19 to 26 nucleotides.

[0188] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV each have a length of 1 to 4 nucleotides, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, the nucleotide sequence III is bound to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is bound to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely reverse complementary, and the second nucleotide sequence refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO: 661 in the target mRNA and having a length equal to that of the nucleotide sequence IV.

[0189] In some embodiments, in the 5'-3' direction, the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide in length, the base of the nucleotide sequence III is U and the base of the nucleotide sequence IV is A, and the length ratio of the sense strand to the antisense strand is 20 / 20; or the nucleotide sequences III and IV are both 2 nucleotides in length, the base sequence of the nucleotide sequence III is AU and the base sequence of the nucleotide sequence IV is AU from the 5' end to the 3' end, and the length ratio of the sense strand to the antisense strand is 21 / 21. or nucleotide sequences III and IV are each 3 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is UAU and the base sequence of nucleotide sequence IV is AUA, and the length ratio of the sense strand to the antisense strand is 22 / 22; or nucleotide sequences III and IV are each 4 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is CUAU and the base sequence of nucleotide sequence IV is AUAG, and the length ratio of the sense strand to the antisense strand is 23 / 23. In some embodiments, the nucleotide sequences III and IV are each 2 nucleotides in length, from the 5' to the 3' end, the base sequence of nucleotide sequence III is AU and the base sequence of nucleotide sequence IV is AU, and the length ratio of the sense strand to the antisense strand is 21 / 21.

[0190] In some embodiments, nucleotide sequence III and nucleotide sequence IV are perfectly reverse complementary, such that given a base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.

[0191] The following description of the nucleotide sequence V, the nucleic acid sequence, the nucleotide modification in the siRNA, and the modified sequence can be applied to any one of the above 1st to 12th siRNAs. That is, unless otherwise specified, the following description of the siRNA should be considered as describing the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA, the sixth siRNA, the seventh siRNA, the eighth siRNA, the ninth siRNA, the tenth siRNA, the eleventh siRNA, and the twelfth siRNA one by one. For example, unless a specific siRNA is specified, "the siRNA further comprises a nucleotide sequence V" means "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA, the fifth siRNA, the sixth siRNA, the seventh siRNA, the eighth siRNA, the ninth siRNA, the tenth siRNA, the eleventh siRNA, or the twelfth siRNA further comprises a nucleotide sequence V."

[0192] In some embodiments, the antisense strand further comprises a nucleotide sequence V, the nucleotide sequence V being 1-3 nucleotides in length and bound to the 3' end of the antisense strand, constituting the 3' overhanging end of the antisense strand. This allows the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure to be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the nucleotide sequence V is 2 nucleotides in length, thereby allowing the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure to be 19 / 21, 21 / 23, or 23 / 25.

[0193] Each nucleotide in the nucleotide sequence V can be any nucleotide, and in order to facilitate synthesis and save synthesis cost, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); or, in order to improve the affinity between the antisense strand of siRNA and target mRNA, the nucleotide sequence V is complementary to the nucleotide at the corresponding position of target mRNA. Thus, in some embodiments, the length ratio of the sense strand and the antisense strand of the siRNA of the present disclosure is 19 / 21 or 21 / 23, and the siRNA of the present disclosure has better target mRNA silencing activity.

[0194] The nucleotide at the corresponding position of the target mRNA refers to a nucleotide or a nucleotide sequence adjacent to the nucleotide sequence of the target mRNA at the 5' end, which is a nucleotide sequence that is substantially or completely reverse complementary to nucleotide sequence II, or a nucleotide sequence that is substantially or completely reverse complementary to the nucleotide sequence consisting of nucleotide sequence II and nucleotide sequence IV.

[0195] In some embodiments, for the first siRNA, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:5, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:6. 5'-GAGAUGAAGUUCAAGAAUZ 3 -3' (SEQ ID NO:5), 5'-Z 4 AUUCUUGAACUUCAUCUCAA-3' (SEQ ID NO:6)

[0196] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:7, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:8. 5'-UUGAGAUGAAGUUCAAGAAUZ 3 -3' (SEQ ID NO: 7), 5'-Z 4 AUUCUUGAACUUCAUCUCAAUG-3' (SEQ ID NO: 8) However, the above Z 4 is the first nucleotide at the 5' end of the antisense strand, and Z 4 is selected from A, U, G or C; Z 3 is Z 4 is a nucleotide complementary to

[0197] In some embodiments, for the second siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:65, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:66. 5'-CAUAACUGGAAUUUGUAAZ 7 -3' (SEQ ID NO: 65), 5'-Z 8 UUACAAAUUCCAGUUAUGUU-3' (SEQ ID NO:66)

[0198] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:67, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:68. 5'-AACAUAACUGGAAUUUGUAAZ 7 -3' (SEQ ID NO: 67), 5'-Z 8 UUACAAAUUCCAGUUAUGUUAC-3' (SEQ ID NO:68) However, the above Z 8 is the first nucleotide at the 5' end of the antisense strand, and Z 8 is selected from A, U, G or C; Z 7 is Z 8 is a nucleotide complementary to

[0199] In some embodiments, for the third siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:125, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:126. 5'-CAUUAUCACAAUUGAGGAZ 11 -3' (SEQ ID NO: 125), 5'-Z 12 UCCUCAAUUGUGAUAAUGGC-3' (SEQ ID NO: 126)

[0200] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:127, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:128. 5'-GCCAUUAUCACAAUUGAGGAZ 11 -3' (SEQ ID NO: 127), 5'-Z 12 UCCUCAAUUGUGAUAAUGGCUG-3' (SEQ ID NO: 128) However, the above Z 12 is the first nucleotide at the 5' end of the antisense strand, and Z 12 is selected from A, U, G or C; Z 11 is Z 12 is a nucleotide complementary to

[0201] In some embodiments, for the fourth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:185, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:186. 5'-GGAUCUCUCUCAGAGUAUZ 15 -3' (SEQ ID NO: 185), 5'-Z 16 AUACUCUGAGAGAGAUCCUG-3' (SEQ ID NO: 186)

[0202] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:187, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:188. 5'-CAGGAUCUCUCUCAGAGUAUZ 15 -3' (SEQ ID NO: 187), 5'-Z 16AUACUCUGAGAGAGAUCCUGGG-3' (SEQ ID NO: 188) However, the above Z 16 is the first nucleotide at the 5' end of the antisense strand, and Z 16 is selected from A, U, G or C; Z 15 is Z 16 is a nucleotide complementary to

[0203] In some embodiments, for the fifth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:245, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:246. 5'-ACAUGGACAACUGCUAUAZ 19 -3' (SEQ ID NO: 245), 5'-Z 20 UAUAGCAGUUGUCCAUGUGG-3' (SEQ ID NO: 246)

[0204] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:247, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:248. 5'-CCACAUGGACAACUGCUAUAZ 19 -3' (SEQ ID NO: 247), 5'-Z 20 UAUAGCAGUUGUCCAUGUGGAA-3' (SEQ ID NO: 248) However, the above Z 20 is the first nucleotide at the 5' end of the antisense strand, and Z 20 is selected from A, U, G or C; Z 19 is Z 20 is a nucleotide complementary to

[0205] In some embodiments, for the sixth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:305, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:306. 5'-UAGCAAGCUCUCAGUAUCZ 23 -3' (SEQ ID NO: 305), 5'-Z 24 GAUACUGAGAGCUUGCUAGG-3' (SEQ ID NO: 306)

[0206] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:307, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:308. 5'-CCUAGCAAGCUCUCAGUAUCZ 23 -3' (SEQ ID NO: 307), 5'-Z 24 GAUACUGAGAGCUUGCUAGGCA-3' (SEQ ID NO: 308) However, the above Z 24 is the first nucleotide at the 5' end of the antisense strand, and Z 24 is selected from A, U, G or C; Z 23 is Z 24 is a nucleotide complementary to

[0207] In some embodiments, for the seventh siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:365, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:366. 5'-AUAAGGUUACUUGUGUUGZ 27 -3' (SEQ ID NO: 365), 5'-Z 28 CAACACAAGUAACCUUAUCC-3' (SEQ ID NO: 366)

[0208] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:367, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:368. 5'-GGAUAAGGUUACUUGUGUUGZ 27 -3' (SEQ ID NO: 367), 5'-Z 28CAACACAAGUAACCUUAUCCUU-3' (SEQ ID NO: 368) However, the above Z 28 is the first nucleotide at the 5' end of the antisense strand, and Z 28 is selected from A, U, G or C; Z 27 is Z 28 is a nucleotide complementary to

[0209] In some embodiments, for the eighth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:425, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:426. 5'-GAAAAUCACCUAUGAAGAZ 31 -3' (SEQ ID NO: 425), 5'-Z 32 UCUUCAUAGGUGAUUUUCAC-3' (SEQ ID NO: 426)

[0210] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:427, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:428. 5'-GUGAAAAUCACCUAUGAAGAZ 31 -3' (SEQ ID NO: 427), 5'-Z 32 UCUUCAUAGGUGAUUUUCACCC-3' (SEQ ID NO: 428) However, the above Z 32 is the first nucleotide at the 5' end of the antisense strand, and Z 32 is selected from A, U, G or C; Z 31 is Z 32 is a nucleotide complementary to

[0211] In some embodiments, for the ninth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:485, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:486. 5'-GAUGCUAUAAAGAACAACZ 35 -3' (SEQ ID NO: 485), 5'-Z 36 GUUGUUCUUUAUAGCAUCCU-3' (SEQ ID NO: 486)

[0212] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:487, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:488. 5'-AGGAUGCUAUAAAGAACAACZ 35 -3' (SEQ ID NO: 487), 5'-Z 36 GUUGUUCUUUAUAGCAUCCUCA-3' (SEQ ID NO: 488) However, the above Z 36 is the first nucleotide at the 5' end of the antisense strand, and Z 36 is selected from A, U, G or C; Z 35 is Z 36 is a nucleotide complementary to

[0213] In some embodiments, for the tenth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:545, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:546. 5'-GAACAACUCCUUUUAUGGZ 39 -3' (SEQ ID NO: 545), 5'-Z 40 CCAUAAAAGGAGUUGUUCUU-3' (SEQ ID NO:546)

[0214] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:547, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:548. 5'-AAGAACAACUCCUUUUAUGGZ 39 -3' (SEQ ID NO: 547), 5'-Z 40CCAUAAAAGGAGUUGUUCUUUA-3' (SEQ ID NO:548) However, the above Z 40 is the first nucleotide at the 5' end of the antisense strand, and Z 40 is selected from A, U, G or C; Z 39 is Z 40 is a nucleotide complementary to

[0215] In some embodiments, for the eleventh siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:605, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:606. 5'-CUUGCUCUGAAGUAGAAAZ 43 -3' (SEQ ID NO: 605), 5'-Z 44 UUUCUACUUCAGAGCAAGCC-3' (SEQ ID NO: 606)

[0216] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:607, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:608. 5'-GGCUUGCUCUGAAGUAGAAAZ 43 -3' (SEQ ID NO: 607), 5'-Z 44 UUUCUACUUCAGAGCAAGCCAC-3' (SEQ ID NO: 608) However, the above Z 44 is the first nucleotide at the 5' end of the antisense strand, and Z 44 is selected from A, U, G or C; Z 43 is Z 44 is a nucleotide complementary to

[0217] In some embodiments, for the twelfth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:665, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:666. 5'-CUUCUUUGCCAUCAAAGAZ 47 -3' (SEQ ID NO: 665), 5'-Z 48 UCUUUGAUGGCAAAGAAGAU-3' (SEQ ID NO: 666)

[0218] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:667, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:668. 5'-AUCUUCUUUGCCAUCAAAGAZ 47 -3' (SEQ ID NO: 667), 5'-Z 48 UCUUUGAUGGCAAAGAAGAUAG-3' (SEQ ID NO: 668) However, the above Z 48 is the first nucleotide at the 5' end of the antisense strand, and Z 48 is selected from A, U, G or C; Z 47 is Z 48 is a nucleotide complementary to

[0219] In some embodiments, the siRNA described in the present disclosure is any one of siXOa1, siXOa2, siXOb1, siXOb2, siXOc1, siXOc2, siXOd1, siXOd2, siXOe1, siXOe2, siXOf1, siXOf2, siXOg1, siXOg2, siXOh1, siXOh2, siXOi1, siXOi2, siXOj1, siXOj2, siXOk1, siXOk2, siXOl1, and siXOl2 as described in Tables 1a-1l.

[0220] As mentioned above, each nucleotide in the siRNA of the present disclosure is independently modified or unmodified nucleotide.In some embodiments, each nucleotide in the siRNA of the present disclosure is unmodified nucleotide.In some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, and these modifications on nucleotide base do not obviously weaken or eliminate the function of the siRNA complex of the present disclosure to suppress XO gene expression.

[0221] In some embodiments, the siRNA of the present disclosure comprises at least one modified nucleotide. In the context of this specification, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced with another group, or a nucleotide having a modified base. The modified nucleotide does not obviously weaken or eliminate the function of siRNA to suppress gene expression. For example, the modified nucleotide disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 may be selected.

[0222] In some embodiments, the sense strand or the antisense strand of the siRNA provided by the present disclosure has at least one nucleotide that is a modified nucleotide and / or at least one phosphate group that is a phosphate group having a modified group. In other words, at least a portion of the phosphate groups and / or ribose groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group having a modified group and / or a ribose group having a modified group.

[0223] In some embodiments, all of the nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

[0224] The inventors of the present disclosure have surprisingly found that the siRNAs described in the present disclosure provide an excellent balance between plasma stability and gene silencing efficiency in animal studies.

[0225] In some embodiments, the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, wherein, from the 5' to the 3' end, at least the 7th, 8th, and 9th nucleotides of nucleotide sequence I are fluoro-modified nucleotides, and from the 5' to the 3' end, at least the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II are fluoro-modified nucleotides.

[0226] In some embodiments, the fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, wherein there are no more than 5 fluoro-modified nucleotides in nucleotide sequence I, and from the 5' end to the 3' end, at least the 7th, 8th and 9th nucleotides of nucleotide sequence I are fluoro-modified nucleotides, there are no more than 7 fluoro-modified nucleotides in nucleotide sequence II, and at least the 2nd, 6th, 14th and 16th nucleotides of nucleotide sequence II are fluoro-modified nucleotides.

[0227] In some embodiments, from the 5' to the 3' end, in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of nucleotide sequence I are fluoro-modified nucleotides and the remaining nucleotides in the sense strand are non-fluoro-modified nucleotides; and from the 5' to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of nucleotide sequence II are fluoro-modified nucleotides and the remaining nucleotides in the antisense strand are non-fluoro-modified nucleotides.

[0228] In the context of this specification, "fluoro-modified nucleotide" refers to a nucleotide having the structure shown in the following formula (7), in which the hydroxy group at the 2' position of the ribose group of the nucleotide is replaced with fluorine. "Non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxy group at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group. In some embodiments, each non-fluoro-modified nucleotide is one independently selected from nucleotides or nucleotide analogs in which the hydroxy group at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group.

[0229] These nucleotides in which the hydroxy group at the 2' position of the ribose group is substituted with a non-fluorine group are known to those skilled in the art, and these nucleotides may be one selected from 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0230] In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe) as shown in formula (8). In some embodiments, the 2'-substituted alkoxy modified nucleotide may be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (9). In some embodiments, the 2'-amino modified nucleotide (2'-NH 2 ) is shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (11).

[0231] [ka]

[0232] A nucleotide analog refers to a group that can substitute for a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, a nucleotide analog can be an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide.

[0233] BNA refers to a constrained or inaccessible nucleotide. BNAs may include five-, six-, or seven-membered bridged structures with a "fixed" C3'-endo sugar puckering. Typically, the bridge is introduced at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, the BNA may be an LNA as shown in formula (12), an ENA as shown in formula (13), a cET BNA as shown in formula (14), or the like.

[0234] [ka]

[0235] An acyclic nucleotide is a nucleotide in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) as shown in formula (15) or a glycerol nucleic acid (GNA) as shown in formula (16).

[0236] [ka] In the above formula (15) and formula (16), R is selected from H, OH, or alkoxy (O-alkyl).

[0237] An isonucleotide refers to a compound in which the position of the base in the ribose ring in a nucleotide is changed. In some embodiments, an isonucleotide may be a compound in which the base is shifted from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (17) or (18).

[0238] [ka] In the compounds of the above formulae (17) and (18), Base represents a nucleic acid base such as A, U, G, C or T, and R is selected from H, OH, F or the above-mentioned non-fluorine groups.

[0239] In some embodiments, the nucleotide analog is one selected from an isonucleotide, an LNA, an ENA, a cET, an UNA, and a GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which in this context refers to a nucleotide in which the 2'-hydroxy group of the ribose group is replaced with methoxy.

[0240] In this context, the terms "fluoro-modified nucleotide", "2'-fluoro-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced with fluorine" and "nucleotide having a 2'-fluoro ribose group" have the same meaning and all refer to a compound having the structure shown in formula (7) in which the 2'-hydroxy group of the nucleotide is replaced with fluorine, and the terms "methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced with methoxy" and "nucleotide having a 2'-methoxy ribose group" have the same meaning and all refer to a compound having the structure shown in formula (8) in which the 2'-hydroxy group of the ribose group of the nucleotide is replaced with methoxy.

[0241] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications: from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence I are fluoro-modified nucleotides, and in the sense strand, the nucleotides at the remaining positions are methoxy-modified nucleotides, and in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluoro-modified nucleotides, and in the antisense strand, the nucleotides at the remaining positions are methoxy-modified nucleotides.

[0242] In some embodiments, the siRNA of the present disclosure is an siRNA having the following modifications: from the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, and the remaining nucleotides in the sense strand of the siRNA are methoxy-modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, and the remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides; Alternatively, from the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides; and from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides; Alternatively, from the 5' to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides; and, from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides.

[0243] In some embodiments, the siRNA provided by the present disclosure is selected from the group consisting of siXOa1-M1, siXOa1-M2, siXOa1-M3, siXOa2-M1, siXOa2-M2, siXOa2-M3, siXOb1-M1, siXOb1-M2, siXOb1-M3, siXOb2-M1, siXOb2-M2, siXOb2-M3, siXOc1-M1, siXOc1-M2, siXOc1-M3, siXOc1-M4, siXOc1-M5, siXOc1-M6, siXOc1-M7, siXOc1-M8, siXOc1-M9, siXOc1-M10, siXOc1-M111, siXOc1-M12, siXOc1-M131, siXOc1-M14, siXOc1-M15, siXOc1-M16, siXOc1-M17, siXOc1-M18, siXOc1-M19, siXOc1-M21, siXOc1-M19, siXOc1-M111, siXOc1-M112, siXOc1-M113, siXOc1-M114, siXOc1-M115, siXOc1-M116, siXOc1-M117, siXOc1-M118, siXOc1-M119, siXOc1-M119, siXOc1-M119, siXOc1-M119, siXO M3, siXOc2-M1, siXOc2-M2, siXOc2-M3, siXOd1-M1, siXOd1-M2, siXOd1-M3, siXOd2-M1, siXOd2-M2, siXOd2-M3, siXO e1-M1, siXOe1-M2, siXOe1-M3, siXOe2-M1, siXOe2-M2, siXOe2-M3, siXOf1-M1, siXOf1-M2, siXOf1-M3, siXOf2-M1, si XOf2-M2, siXOf2-M3, siXOg1-M1, siXOg1-M2, siXOg1-M3, siXOg2-M1, siXOg2-M2, siXOg2-M3, siXOh1-M1, siXOh1-M2 , siXOh1-M3, siXOh2-M1, siXOh2-M2, siXOh2-M3, siXOi1-M1, siXOi1-M2, siXOi1-M3, siXOi2-M1, siXOi2-M2, siXOi2 -M3, siXOj1-M1, siXOj1-M2, siXOj1-M3, siXOj2-M1, siXOj2-M2, siXOj2-M3, siXOk1-M1, siXOk1-M2, siXOk1-M3, siX It is any one of Ok2-M1, siXOk2-M2, siXOk2-M3, siXOl1-M1, siXOl1-M2, siXOl1-M3, siXOl2-M1, siXOl2-M2, and siXOl2-M3.

[0244] The siRNA having the above modification is not only low-cost, but also can make nucleic acid difficult to be cut by ribonuclease in blood, thereby improving the stability of nucleic acid and further enhancing the property of nucleic acid resistance to nuclease hydrolysis.In addition, the above modified siRNA has high target mRNA suppression activity.

[0245] In some embodiments, in the sense strand and antisense strand of the siRNA provided by the present disclosure, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand is a phosphate ester group having a modified group.In some embodiments, the phosphate ester group having a modified group is a thiophosphate ester group in which at least one oxygen atom of the phosphodiester bond in the phosphate ester group is replaced with a sulfur atom.In some embodiments, the phosphate ester group having a modified group is a thiophosphate ester group having the structure shown in formula (1).

[0246] [ka]

[0247] Such modifications stabilize the double-stranded structure of siRNA and enable high specificity and high affinity base pairing to be maintained.

[0248] In some embodiments, in the siRNA provided by the present disclosure, the thiophosphate group is present at least one of the following positions: between the first and second nucleotides at any one end of the sense strand or antisense strand, between the second and third nucleotides at any one end of the sense strand or antisense strand, or any combination thereof. In some embodiments, the thiophosphate group is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the thiophosphate group is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the thiophosphate group is present at at least one of the following positions: Between the first and second nucleotides from the 5' end of the sense strand, Between the second and third nucleotides from the 5' end of the sense strand, Between the first and second nucleotides from the 3' end of the sense strand, Between the second nucleotide and the third nucleotide from the 3' end of the sense strand, Between the first and second nucleotides from the 5' end of the antisense strand, Between the second and third nucleotides from the 5' end of the antisense strand, Between the first and second nucleotides from the 3' end of the antisense strand, and Between the second and third nucleotides from the 3' end of the antisense strand.

[0249] In some embodiments, the siRNA provided by the present disclosure is selected from the group consisting of siXOa1-M1S, siXOa1-M2S, siXOa1-M3S, siXOa2-M1S, siXOa2-M2S, siXOa2-M3S, siXOb1-M1S, siXOb1-M2S, siXOb1-M3S, siXOb2-M1S, siXOb2-M2S, siXOb2-M3S, siXOc1-M1S, siXOc1-M2S, and siXOc1-M3S, as set forth in Tables 1a-1l. , siXOc2-M1S, siXOc2-M2S, siXOc2-M3S, siXOd1-M1S, siXOd1-M2S, siXOd1-M3S, siXOd2-M1S, siXOd2-M2S, siXOd2-M3S, siXOe1 -M1S, siXOe1-M2S, siXOe1-M3S, siXOe2-M1S, siXOe2-M2S, siXOe2-M3S, siXOf1-M1S, siXOf1-M2S, siXOf1-M3S, siXOf2-M1S, si XOf2-M2S, siXOf2-M3S, siXOg1-M1S, siXOg1-M2S, siXOg1-M3S, siXOg2-M1S, siXOg2-M2S, siXOg2-M3S, siXOh1-M1S, siXOh1-M2 S, siXOh1-M3S, siXOh2-M1S, siXOh2-M2S, siXOh2-M3S, XOi1-M1S, siXOi1-M2S, siXOi1-M3S, siXOi2-M1S, siXOi2-M2S, siXOi2- M3S, siXOj1-M1S, siXOj1-M2S, siXOj1-M3S, siXOj2-M1S, siXOj2-M2S, siXOj2-M3S, siXOk1-M1S, siXOk1-M2S, siXOk1-M3S, siXOk2-M1S, siXOk2-M2S, siXOk2-M3S, siXOl1-M1S, siXOl1-M2S, siXOl1-M3S, siXOl2-M1S, siXOl2-M2 and siXOl2-M3S.

[0250] In some embodiments, the 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.

[0251] The conventional 5'-phosphate nucleotides or 5'-phosphate analogue modified nucleotides are known to those skilled in the art. For example, the 5'-phosphate nucleotide may have the following structure:

[0252] [ka]

[0253] Furthermore, for example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four types of 5'-phosphate analog modified nucleotides:

[0254] [ka] In the formula, R is selected from H, OH, methoxy, and fluorine, and Base represents a nucleobase and is selected from A, U, C, G, or T.

[0255] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), and the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate (E-VP)) modification as shown in formula (3), or a thiophosphate modified nucleotide as shown in formula (5).

[0256] In some embodiments, the siRNA provided by the present disclosure is selected from the group consisting of siXOa1-M1P1, siXOa1-M2P1, siXOa1-M3P1, siXOa2-M1P1, siXOa2-M2P1, siXOa2-M3P1, siXOa1-M1SP1, siXOa1-M2SP1, siXOa1-M3SP1, siXOa2-M1SP1, siXOa2-M2SP1, siXOa2-M3SP1, siXOb1-M1P1, siXOb1-M2P1, siXOb1-M3P1, siXOb2-M1P1, siXOb2-M1P1, siXOb2-M2P1, siXOb2-M3P1, siXOa1-M1SP1, siXOa1-M3SP1, siXOa2-M1SP1, siXOa2-M2SP1, siXOa2-M3SP1, siXOb1-M1P1, siXOb1-M2P1, siXOb1-M3P1, siXOb2-M1P1, siXOb2-M2P1, siXOb2 ... Ob2-M2P1, siXOb2-M3P1, siXOb1-M1SP1, siXOb1-M2SP1, siXOb1-M3SP1, siXOb2-M1SP1, siXOb2-M2SP1, siXOb2-M3SP1, siXOc1-M1P1, siXOc1-M2P1, s iXOc1-M3P1, siXOc2-M1P1, siXOc2-M2P1, siXOc2-M3P1, siXOc1-M1SP1, siXOc1-M2SP1, siXOc1-M3SP1, siXOc2-M1SP1, siXOc2-M2SP1, siXOc2-M3SP1, siXOd1-M1P1, siXOd1-M2P1, siXOd1-M3P1, siXOd2-M1P1, siXOd2-M2P1, siXOd2-M3P1, siXOd1-M1SP1, siXOd1-M2SP1, siXOd1-M3SP1, siXOd2-M1SP1, siXOd2-M2SP1, siXOd2-M3SP1, siXOe1-M1P1, siXOe1-M2P1, siXOe1-M3P1, siXOe2-M1P1, siXOe2-M2P1, siXOe2-M3P1, siXOe1-M1SP1, siXOe1-M2SP1, s iXOe1-M3SP1, siXOe2-M1SP1, siXOe2-M2SP1, siXOe2-M3SP1, siXOf1-M1P1, siXOf1-M2P1, siXOf1-M3P1, siXOf2-M1P1, siXOf2-M2P1, siXOf2-M3P1, s iXOf1-M1SP1, siXOf1-M2SP1, siXOf1-M3SP1, siXOf2-M1SP1, siXOf2-M2SP1, siXOf2-M3SP1, siXOg1-M1P1, siXOg1-M2P1, siXOg1-M3P1, siXOg2-M1P1,siXOg2-M2P1, siXOg2-M3P1, siXOg1-M1SP1, siXOg1-M2SP1, siXOg1-M3SP1, siXOg2-M1SP1, siXOg2-M2SP1, siXOg2-M3SP1, siXOh 1-M1P1, siXOh1-M2P1, siXOh1-M3P1, siXOh2-M1P1, siXOh2-M2P1, siXOh2-M3P1, siXOh1-M1SP1, siXOh1-M2SP1, siXOh1-M3SP1, si XOh2-M1SP1,siXOh2-M2SP1,siXOh2-M3SP1,XOi1-M1P1,siXOi1-M2P1,siXOi1-M3P1,siXOi2-M1P1,siXOi2-M2P1,siXOi2-M3P1, siXOi1-M1SP1, siXOi1-M2SP1, siXOi1-M3SP1, siXOi2-M1SP1, siXOi2-M2SP1, siXOi2-M3SP1, siXOj1-M1P1, siXOj1-M2P1, siXOj1 -M3P1, siXOj2-M1P1, siXOj2-M2P1, siXOj2-M3P1, siXOk1-M1P1, siXOk1-M2P1, siXOk1-M3P1, siXOk2-M1P1, siXOk2-M2P1, siXOk 2-M3P1, siXOl1-M1P1, siXOl1-M2P1, siXOl1-M3P1, siXOl2-M1P1, siXOl2-M2P1, siXOl2-M3P1, siXOj1-M1SP1, siXOj1-M2SP1, siX It is any one of Oj1-M3SP1, siXOj2-M1SP1, siXOj2-M2SP1, siXOj2-M3SP1, siXOk1-M1SP1, siXOk1-M2SP1, siXOk1-M3SP1, siXOk2-M1SP1, siXOk2-M2SP1, siXOk2-M3SP1, siXOl1-M1SP1, siXOl1-M2SP1, siXOl1-M3SP1, siXOl2-M1SP1, siXOl2-M2SP1 and siXOl2-M3SP1.

[0257] The inventors of the present disclosure have unexpectedly found that the above siRNAs provided by the present disclosure not only have significantly improved plasma and lysosomal stability, but also have high target mRNA silencing activity.

[0258] The siRNA provided by the present disclosure can be obtained by the usual siRNA preparation methods in the field (for example, solid-phase synthesis method and liquid-phase synthesis method).Here, solid-phase synthesis has already been commercialized as a customization service.The modified nucleotide group can be introduced into the siRNA described in the present disclosure by using the nucleoside monomer having the corresponding modification, and the method of preparing the nucleoside monomer having the corresponding modification and the method of introducing the modified nucleotide group into the siRNA are also well known to those skilled in the art.

[0259] <Drug Composition> The present disclosure provides a pharmaceutical composition comprising the above-mentioned siRNA as an active ingredient and a pharma- ceutically acceptable carrier.

[0260] The pharma- ceutical acceptable carrier may be a carrier commonly used in the field of siRNA administration, for example, magnetic nanoparticles (e.g., Fe 3 O 4 or Fe 2 O 3The nanoparticles may include, but are not limited to, one or more of nanoparticles based on PEG-100, carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA), and derivatives thereof.

[0261] There is no particular requirement for the content of siRNA and pharma- ceutically acceptable carrier in the drug composition, but each component may have a normal content. In some embodiments, the weight ratio of siRNA to pharma-ceutically acceptable carrier may be 1:(1-500). In some embodiments, the weight ratio is 1:(1-50).

[0262] In some embodiments, the drug composition may include other pharma- ceutically acceptable additives, which may be one or more of various preparations or compounds commonly used in the art. For example, the other pharma-ceutically acceptable additives may include at least one of a pH buffer, a protective agent, and an osmolality adjusting agent.

[0263] The pH buffer solution may be a tris(hydroxymethyl) aminomethane hydrochloride buffer having a pH of 7.5 to 8.5 and / or a phosphate buffer solution having a pH of 5.5 to 8.5, for example, a phosphate buffer solution having a pH of 5.5 to 8.5.

[0264] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the drug composition, the content of the protective agent may be 0.01 to 30% by weight.

[0265] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is determined so that the osmotic pressure of the drug composition is 200-700 milliosmoles / kilogram (mOsm / kg). Depending on the desired osmotic pressure, a person skilled in the art can easily determine the content of the osmotic pressure regulator.

[0266] In some embodiments, the pharmaceutical composition may be a liquid formulation such as an injection solution, or may be a lyophilized powder injection that is mixed with a liquid additive at the time of administration to form a liquid formulation. The liquid formulation may be used for, but is not limited to, subcutaneous, intramuscular, or intravenous administration, and may also be administered to the lungs by aerosolization, or to other organ tissues (e.g., the liver) through the lungs by aerosolization. In some embodiments, the pharmaceutical composition is used for intravenous administration.

[0267] In some embodiments, the drug composition may be in the form of a liposome formulation. In some embodiments, the pharma- ceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as organic amine), an auxiliary lipid, and / or a PEGylated (polyethylene glycol) lipid. Here, the organic amine, the auxiliary lipid, and the PEGylated lipid may be one or more selected from the amine-containing transfection compound or its pharma- ceutically acceptable salt or derivative, the auxiliary lipid, and the PEGylated lipid described in CN103380113A (incorporated herein in its entirety by reference).

[0268] In some embodiments, the organic amine may be a compound represented by formula (201) described in CN103380113A or a pharma- ceutically acceptable salt thereof.

[0269] [ka] During the ceremony, each X 101 Or X 102 are each independently O, S, NA, or CA, and A is hydrogen or C 1 -C 20 is a hydrocarbon chain, Each Y 101 or Z 101 are each independently C=O, C=S, S=O, CH-OH or SO 2 and Each R 101 , R 102 , R 103 , R 104 , R 105 , R 106 or R 107 each independently represents hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, or a substituted or unsubstituted, branched or straight-chain heteroaryl group; x is an integer from 1 to 10; When n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1, where m=p=0, R 102 is hydrogen, When at least one of n or m is 2, R 103 and the nitrogen in formula (201) form a structure shown in formula (202) or formula (203).

[0270] [ka] In the formula, g, e, and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each * N represents a nitrogen atom in formula (201).

[0271] In some embodiments, R 103 is a polyamine. In another embodiment, R 103 is a ketal. In some embodiments, R in formula (201) 101 and R 102 each is independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl, said alkyl or alkenyl having 3 to about 20 carbon atoms, e.g., 8 to about 18 carbon atoms, and 0 to 4 double bonds, e.g., 0 to 2 double bonds.

[0272] In some embodiments, when n and m each independently have a value of 1 or 3, R 103 may be any one of the following formulas (204) to (213).

[0273] [ka] In formulae (204) to (213), g, e, and f each independently represent an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, * is R 103 and the nitrogen atom in formula (201), and *Each H on position may be substituted to bond with a nitrogen atom in formula (201).

[0274] Compounds of formula (201) may be prepared as described in CN103380113A.

[0275] In some embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215).

[0276] [ka]

[0277] the co-lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative; The PEGylated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0278] In some embodiments, the molar ratio of the organic amine, the co-lipid, and the PEGylated lipid in the drug composition is (19.7-80):(19.7-80):(0.3-50), and may be, for example, (50-70):(20-40):(3-20).

[0279] In some embodiments, the drug composition particles formed by the siRNA of the present disclosure and the amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, e.g., the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0280] In some embodiments, in the drug composition formed by the siRNA of the present disclosure and the amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the siRNA to the total lipid (e.g., organic amine, auxiliary lipid, and / or PEGylated lipid) is within the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12, or about 1:6 to about 1:10, for example, the weight ratio of the siRNA of the present disclosure to the total lipid is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

[0281] In some embodiments, the pharmaceutical composition may be marketed with each component being independent, or may be used as a liquid formulation.In some embodiments, the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutical acceptable carrier may be prepared according to various known methods, and the siRNA provided by the present disclosure may be used instead of conventional siRNA.In some embodiments, the pharmaceutical composition may be prepared according to the following method.

[0282] The organic amine, auxiliary lipid and PEGylated lipid are suspended in alcohol in the above molar ratio and mixed uniformly to obtain a lipid solution. The amount of alcohol is determined so that the total mass concentration of the obtained lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol is one or more selected from pharma- ceutically acceptable alcohols, such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, and other alcohols that are liquid at around room temperature, and may be, for example, ethanol.

[0283] The siRNA provided by the present disclosure is dissolved in a buffer salt solution to obtain an siRNA aqueous solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, and may be, for example, 0.1 to 0.2 M. The pH of the buffer salt solution is adjusted to 4.0 to 5.5, and may be, for example, 5.0 to 5.2. The amount of the buffer salt solution is determined so that the concentration of the siRNA is 0.6 mg / mL or less, and may be, for example, 0.2 to 0.4 mg / mL. The buffer salt is one or more selected from soluble acetates and soluble citrates, and may be, for example, sodium acetate and / or potassium acetate.

[0284] After mixing the lipid solution and the siRNA aqueous solution, the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome formulation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(2-5), and may be, for example, 1:4.

[0285] The cultured liposome preparation is concentrated or diluted, impurities are removed, and bacteria are sterilized to obtain a drug composition provided by the present disclosure. Its physicochemical parameters are pH 6.5-8, encapsulation efficiency 80% or more, particle size 40-200 nm, polydispersity index 0.30 or less, and osmotic pressure 250-400 mOsm / kg. For example, the physicochemical parameters may be pH 7.2-7.6, encapsulation efficiency 90% or more, particle size 60-100 nm, polydispersity index 0.20 or less, and osmotic pressure 300-400 mOsm / kg.

[0286] Here, concentration or dilution may be performed before, after, or simultaneously with the removal of impurities. As a method for removing impurities, various conventional methods may be employed. For example, a tangential flow system or a hollow fiber column may be used, and ultrafiltration may be performed under the condition of 100 KDa. The ultrafiltration exchange solution may be a phosphate buffer (PBS) with a pH of 7.4. As a sterilization method, various conventional methods may be employed. For example, filtration through a 0.22 μm filter may be used for sterilization.

[0287] <siRNA complex> The present disclosure provides an siRNA complex comprising the above siRNA and a complex group that is complexed and bound to the siRNA.

[0288] Generally, the complex group includes at least one pharmaceutically acceptable target group and an optional linker, and the siRNA, the linker, and the target group are sequentially bound. In some embodiments, the number of the target groups is 1 to 6. In some embodiments, the number of the target groups is 2 to 4. The siRNA molecule may be complexed with the complex group non-covalently or covalently. For example, it may be covalently complexed with the complex group. The complex site between the siRNA and the complex group may be at the 3'-end or 5'-end of the sense strand of the siRNA, at the 5'-end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the complex site between the siRNA and the complex group is at the 3'-terminal of the sense strand of the siRNA.

[0289] In some embodiments, the conjugated group may be bound to the phosphate group, 2'-hydroxy group, or base of the nucleotide. In some embodiments, the conjugated group may be bound to the 3'-hydroxy group, in which case the nucleotides are bound by a 2'-5' phosphodiester bond. When the conjugated group is bound to the end of the siRNA chain, it is usually bound to the phosphate group of the nucleotide, and when it is bound to the internal sequence of the siRNA, it is usually bound to the ribose sugar ring or base. For various binding methods, see Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology,2015,10 (5): 1181-7.

[0290] In some embodiments, the siRNA and the conjugation group can be linked by acid-labile or reducible chemical bonds, and these chemical bonds can be degraded in the acidic environment of cell endosome, and the siRNA can be released.For non-degradable conjugation methods, the conjugation group is bound to the sense strand of siRNA, so that the influence of conjugation on siRNA activity can be minimized.

[0291] In some embodiments, the pharma- ceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the disclosure of which is incorporated herein by reference in its entirety.

[0292] In some embodiments, the pharma- ceutically acceptable targeting group may be one or more selected from lipophilic molecules such as cholesterol, bile acids, vitamins (e.g., tocopherol), lipid molecules of different chain lengths, polymers such as polyethylene glycol, polypeptides such as membrane-permeable peptides, aptamers, antibodies, quantum dots, sugars such as lactose, polylactose, mannose, galactose, and N-acetylgalactosamine (GalNAc), folate, and ligands formed by targeting molecules or derivatives thereof, such as asialoglycoproteins, asialoglycoresidues, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, and receptor ligands expressed in hepatic parenchymal cells, such as transferrin.

[0293] In some embodiments, each of the ligands is independently selected from ligands that can bind to cell surface receptors. In some embodiments, at least one ligand is a ligand that can bind to a receptor on the surface of hepatocytes. In some embodiments, at least one ligand is a ligand that can bind to a receptor on the surface of mammalian hepatocytes. In some embodiments, at least one ligand is a ligand that can bind to a receptor on the surface of human hepatocytes. In some embodiments, at least one ligand is a ligand that can bind to asialoglycoprotein receptor (ASGPR) on the surface of liver. These types of ligands are known to those skilled in the art, and their function is generally to bind to a specific receptor on the surface of target cells and mediate the delivery of siRNA bound to the ligand to the target cells.

[0294] In some embodiments, the pharma- ceutically acceptable targeting group may be any one of the ligands that bind to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0295] In some embodiments, at least one ligand is a sugar. In some embodiments, each of the ligands is a sugar. In some embodiments, at least one of the ligands is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one of the ligands is a modified sugar. In some embodiments, each of the ligands is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one of the ligands is selected from the group consisting of glucose and derivatives thereof, mannan and derivatives thereof, galactose and derivatives thereof, xylose and derivatives thereof, ribose and derivatives thereof, fucose and derivatives thereof, lactose and derivatives thereof, maltose and derivatives thereof, arabinose and derivatives thereof, fructose and derivatives thereof, and sialic acid.

[0296] In some embodiments, each of the ligands is D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, N- Isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl The ligands may be independently selected from ethyl 2,3,4-tris-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-alonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other options for the ligands may be found, for example, in CN105378082A, the disclosure of which is incorporated herein by reference in its entirety.

[0297] In some embodiments, the pharma- ceutically acceptable targeting group in the siRNA complex may be galactose or N-acetylgalactosamine, and the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, or tetravalent. It should be understood that monovalent, divalent, trivalent, and tetravalent herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA complex formed from the siRNA molecule and the conjugated group comprising the galactose or N-acetylgalactosamine molecule as the targeting group being 1:1, 1:2, 1:3, or 1:4, respectively. In some embodiments, the pharma- ceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to the conjugated group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in the present disclosure is conjugated to the conjugated group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0298] Targeting group can be linked to siRNA molecule via suitable linker, and those skilled in the art can select suitable linker according to the specific type of targeting group.For these linkers, types of targeting group and methods of linking to siRNA, reference can be made to the disclosure of WO2015006740A2, the contents of which are incorporated herein by reference in their entirety.

[0299] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be the structure shown in formula (301):

[0300] [ka] During the ceremony, k is an integer from 1 to 3; L A is a chain portion having an amide bond and having a structure represented by formula (302), Ahas one of the target groups and one of the L C It is bonded to the moiety via an ether bond.

[0301] [ka] L B is a chain portion containing an N-acylpyrrolidine having a structure represented by formula (303), the chain portion having a carbonyl group at one end thereof, C It has an oxygen group at its other end and is bonded to the siRNA via a phosphate bond.

[0302] [ka] L C is a divalent to tetravalent linker group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane, C Each of the L's is bonded to an oxygen atom via an ether bond. A and is bonded to the L moiety via an amide bond via a nitrogen atom. B The part is connected to the

[0303] In some embodiments, n=3 and L C When -(L) is a tetravalent linker group based on trihydroxymethylaminomethane, -(L) A ) 3 Trihydroxymethylaminomethane-L B The structure of the siRNA complex formed by binding an N-acetylgalactosamine molecule to an siRNA molecule by - is shown in formula (304) below.

[0304] [ka] In the formula, the double helix structure represents the siRNA.

[0305] Similarly, the conjugation site between the siRNA and the conjugation group may be at the 3' or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in an internal sequence of the siRNA.

[0306] In some embodiments, the 3' end of the sense strand of the siRNA described in the present disclosure is linked to a linker-(L A ) 3 Trihydroxymethylaminomethane-L B - is covalently conjugated to three N-acetylgalactosamine (GalNAc) molecules by the siRNA complex having a structure represented by the following formula (305) (hereinafter, (GalNAc) 3 -siRNA) is obtained.

[0307] [ka] wherein the double helix structure represents the siRNA, and the linker is attached to the 3' end of the sense strand of the siRNA.

[0308] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be the structure shown in formula (306):

[0309] [ka] During the ceremony, l is an integer from 0 to 3; * represents the site on the linker that is attached to the targeting group via an ether bond, # represents the site in the linker that is bound to the siRNA via a phosphate bond.

[0310] In some embodiments, when l=2, the siRNA complex has the structure shown in formula (307).

[0311] [ka] wherein the double helix structure represents the siRNA, and the linker is attached to the 3' end of the sense strand of the siRNA.

[0312] The siRNA complex may be synthesized by a method already described in detail in the prior art. For example, WO2015006740A2 describes in detail the method for preparing multiple types of siRNA complex. The siRNA complex of the present disclosure is obtained by a method well known to those skilled in the art. For example, WO2014025805A1 describes the method for preparing the structure shown in formula (305), and Rajeev et al. described the method for preparing the structure shown in formula (307) in ChemBioChem 2015,16,903-908.

[0313] In some embodiments, the siRNA complex has the structure shown in formula (308):

[0314] [ka] During the ceremony, n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4, m1, m2, and m3 are independently an integer selected from 2 to 10; R 10 , R 11 , R 12 , R 13 , R 14 or R 15 are each independently H or C 1 -C 10 Alkyl group, C 1 -C 10 Halogenated alkyl groups and C 1 -C 10 alkoxy groups; R 3 is a group having the structure shown in formula A59.

[0315] [ka] In the formula, E 1 is OH, SH or BH 2 and Nu is an siRNA of the disclosure.

[0316] R 2 is a straight chain alkylene group 1 to 20 carbon atoms in length, where one or more of the carbon atoms is C(O), NH, O, S, CH=N, S(O) 2 , C 2 -C 10 Alkenylene group, C 2 -C 10 Alkynylene group, C 6 -C 10 Arylene group, C 3 -C 18 Heterocyclylene groups and C 5 -C 10 heteroarylene groups; R 2 is C 1 -C 10 Alkyl group, C 6 -C 10 Aryl group, C 5 -C 10 Heteroaryl groups, C 1 -C 10 Halogenated alkyl groups, -OC 1 -C 10 Alkyl group, -OC 1 -C 10 Alkylphenyl group, -C 1 -C 10 Alkyl-OH, -OC 1 -C 10 Halogenated alkyl groups, -SC 1 -C 10 Alkyl group, -SC 1 -C 10 Alkylphenyl group, -C 1 -C 10 Alkyl-SH, -SC 1 -C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH 2 , -C 1 -C 10 Alkyl-NH2 , -N(C 1 -C 10 Alkyl group)(C 1 -C 10 alkyl group), -NH(C 1 -C 10 alkyl group), N(C 1 -C 10 Alkyl group)(C 1 -C 10 alkylphenyl group), NH(C 1 -C 10 alkylphenyl group), cyano group, nitro group, -CO 2 H, -C(O)O(C 1 -C 10 alkyl group), -CON(C 1 -C 10 Alkyl group)(C 1 -C 10 alkyl group), -CONH(C 1 -C 10 alkyl group), -CONH 2 , -NHC(O)(C 1 -C 10 alkyl group), -NHC(O)(phenyl group), -N(C 1 -C 10 Alkyl group)C(O)(C 1 -C 10 alkyl group), -N(C 1 -C 10 alkyl group)C(O)(phenyl group), -C(O)C 1 -C 10 Alkyl group, -C(O)C 1 -C 10 Alkylphenyl group, -C(O)C 1 -C 10 Haloalkyl groups, -OC(O)C 1 -C 10 Alkyl group, -SO 2 (C 1 -C 10 Alkyl group), -SO 2 (phenyl group), -SO 2 (C 1 -C 10 Halogenated alkyl group), -SO 2 NH 2 , -SO 2 NH(C1 -C 10 Alkyl group), -SO 2 NH (phenyl group), -NHSO 2 (C 1 -C 10 alkyl group), -NHSO 2 (phenyl group) and -NHSO 2 (C 1 -C 10 halogenated alkyl groups), Each L 1 is a straight chain alkylene group of 1 to 70 carbon atoms in length, where one or more of the carbon atoms is C(O), NH, O, S, CH=N, S(O) 2 , C 2 -C 10 Alkenylene group, C 2 -C 10 Alkynylene group, C 6 -C 10 Arylene group, C 3 -C 18 Heterocyclylene group and C 5 -C 10 heteroarylene groups; L 1 is C 1 -C 10 Alkyl group, C 6 -C 10 Aryl group, C 5 -C 10 Heteroaryl groups, C 1 -C 10 Halogenated alkyl groups, -OC 1 -C 10 Alkyl group, -OC 1 -C 10 Alkylphenyl group, -C 1 -C 10 Alkyl-OH, -OC 1 -C 10 Halogenated alkyl groups, -SC 1 -C 10 Alkyl group, -SC 1 -C 10 Alkylphenyl group, -C 1 -C 10 Alkyl-SH, -SC 1-C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH 2 , -C 1 -C 10 Alkyl-NH 2 , -N(C 1 -C 10 Alkyl group)(C 1 -C 10 alkyl group), -NH(C 1 -C 10 alkyl group), N(C 1 -C 10 Alkyl group)(C 1 -C 10 alkylphenyl group), NH(C 1 -C 10 alkylphenyl group), cyano group, nitro group, -CO 2 H, -C(O)O(C 1 -C 10 alkyl group), -CON(C 1 -C 10 Alkyl group)(C 1 -C 10 alkyl group), -CONH(C 1 -C 10 alkyl group), -CONH 2 , -NHC(O)(C 1 -C 10 alkyl group), -NHC(O)(phenyl group), -N(C 1 -C 10 Alkyl group)C(O)(C 1 -C 10 alkyl group), -N(C 1 -C 10 alkyl group)C(O)(phenyl group), -C(O)C 1 -C 10 Alkyl group, -C(O)C 1 -C 10 Alkylphenyl group, -C(O)C 1 -C 10 Haloalkyl groups, -OC(O)C 1 -C 10 Alkyl group, -SO 2 (C 1 -C 10 Alkyl group), -SO 2 (phenyl group), -SO 2(C 1 -C 10 Halogenated alkyl group), -SO 2 NH 2 , -SO 2 NH(C 1 -C 10 Alkyl group), -SO 2 NH (phenyl group), -NHSO 2 (C 1 -C 10 alkyl group), -NHSO 2 (phenyl group) and -NHSO 2 (C 1 -C 10 The alkyl group may optionally have one or more substituents selected from the group consisting of halogenated alkyl groups.

[0317] In some embodiments, L 1 may be selected from the group consisting of groups A1 to A26 or any combination thereof, where the structures and definitions of A1 to A26 are as follows:

[0318] [ka] In the formula, j1 is an integer from 1 to 20, and j2 is an integer from 1 to 20. R' is C 1 -C 10 is an alkyl group, Ra is selected from the group consisting of groups of formulae A27-A45 or any combination thereof.

[0319] [ka] Rb is C 1 -C 10 is an alkyl group, JPEG0007610849000023.jpg12124 represents the site to which the group is covalently attached.

[0320] For convenience, L 1is defined as a linear alkylene group, it will be understood by those skilled in the art that, for example, amines and alkenyl groups resulting from the above-mentioned replacement and / or substitution may not be linear or may have different names. For purposes of this disclosure, L 1 The length of is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring obtained by replacing a carbon atom of the linear alkylene (e.g., heterocyclylene or heteroarylene) is considered to be one atom.

[0321] M 1 represents a targeting group, the definition and selectable range of which are the same as those of the targeting group described above. 1 is one independently selected from ligands that have affinity for the asialoglycoprotein receptor on the surface of mammalian hepatocytes.

[0322] M 1 is a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 may be an integer from 1 to 3, n3 may be an integer from 0 to 4, and M 1 Ensure that the number of targeting groups is at least 2. In some embodiments, n1+n3≧2, such that M 1 The number of target groups is at least 3, and M 1 This allows the targeting group to more easily bind to the asialoglycoprotein receptor on the liver surface, and further promotes the uptake of the siRNA complex into cells by endocytosis (intracellular uptake). 1 When the number of target groups is three or more, M 1 Since the improvement of the ease of binding between the targeting group and the asialoglycoprotein receptor on the liver surface is not clear, in consideration of various aspects such as ease of synthesis, structure / process cost, and delivery efficiency, in some embodiments, n1 is an integer of 1 or 2, n3 is an integer of 0 or 1, and n1+n3=2 or 3.

[0323] In some embodiments, when m1, m2, or m3 is independently selected from an integer of 2 to 10, a plurality of M 1 The spatial position between the target groups is M 1 The targeting group can be adapted to bind to the asialoglycoprotein receptor on the liver surface. To simplify, synthesize, and / or reduce costs of the siRNA complexes provided by the present disclosure, in some embodiments, m1, m2, and m3 are each independently an integer from 2 to 5, and in some embodiments, m1=m2=m3.

[0324] R 10 , R 11 , R 12 , R 13 , R 14 or R 15 But, H, C 1 -C 10 Alkyl group, C 1 -C 10 Halogenated alkyl groups and C 1 -C 10 It will be understood by those skilled in the art that when R is one independently selected from the group consisting of alkoxy groups, the objectives of the present disclosure can be achieved without changing the properties of the siRNA complex of the present disclosure. 10 , R 11 , R 12 , R 13 , R 14 or R 15 are each independently selected from H, a methyl group, or an ethyl group. 10 , R 11 , R 12 , R 13 , R 14 and R 15 are both H.

[0325] R 3 is a group having the structure shown in formula A59, where E 1 is OH, SH or BH 2 In consideration of the availability of preparation raw materials, in some embodiments, E 1 is OH or SH.

[0326] R 2 is selected to achieve a bond between the N atom on the nitrogen-containing backbone and A59. In the context of this specification, a "nitrogen-containing backbone" is defined as R 10 , R 11 , R 12 , R 13 , R 14 and R 15 It refers to a chain structure in which the carbon atom to which R is bonded and the N atom are bonded to each other. 2 may be any linker group capable of linking the A59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, when preparing the siRNA complex shown in formula (308) by a solid phase synthesis process, R 2 The group includes a moiety that is bonded to an N atom on the nitrogen-containing backbone and an R 3 In some embodiments, both the moiety bonded to the P atom in R 2 The site bonded to the N atom on the nitrogen-containing skeleton in the 3 The moiety bonded to the P atom on forms a phosphate bond with the P atom, and in some embodiments, R 2 may be B5, B6, B5' or B6'.

[0327] [ka] During the ceremony, JPEG0007610849000025.jpg12136 represents the site to which the group is covalently attached.

[0328] q 2 The value of q may range from 1 to 10, and in some embodiments, 2 is an integer from 1 to 5.

[0329] L 1 is M 1It serves to link the targeting group to the N on the nitrogen-containing backbone and provide liver targeting functionality to the siRNA complex shown in formula (308). In some embodiments, L 1 is a combination of one or more bonds selected from the groups of formulae A1 to A26. 1 is a combination of one or more bonds selected from A1, A4, A5, A6, A8, A10, A11, and A13. 1 is a combination of at least two bonds selected from A1, A4, A8, A10, and A11. In some embodiments, L 1 is a combination of at least two bonds selected from A1, A8, and A10.

[0330] In some embodiments, L 1 The length of L may be 3 to 25 atoms, 3 to 20 atoms, 4 to 15 atoms, or 5 to 12 atoms. 1 The lengths are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, and 60 atoms.

[0331] In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R' is C 1 -C 4 R is an alkyl group, and in some embodiments, R' is one of a methyl group, an ethyl group, and an isopropyl group. R is one of A27, A28, A29, A30, and A31, and in some embodiments, R is A27 or A28. R is C 1 -C 5In some embodiments, j1, j2, R′, Ra, and Rb in Formulae A1 to A26 are each selected to provide one of M, M, R′, R′, and Rb. 1 The target group is bonded to the N atom on the nitrogen-containing backbone, and M 1 The spatial position between the target groups is M 1 The targeting group is further adapted for binding to the asialoglycoprotein receptor on the liver surface.

[0332] In some embodiments, the siRNA complex has a structure as shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422).

[0333] [ka] JPEG0007610849000027.jpg205132JPEG0007610849000028.jpg206131JPEG0007610849000029.jpg194120 JPEG0007610849000030.jpg233118JPEG0007610849000031.jpg214115JPEG0007610849000032.jpg162121

[0334] In some embodiments, the P atom in formula A59 may be attached to any possible position in the siRNA sequence, for example, the P atom in formula A59 may be attached to any one nucleotide of the sense strand or antisense strand of the siRNA, and in some embodiments, the P atom in formula A59 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is attached to the end of the sense strand or antisense strand of the siRNA, and in some embodiments, the P atom in formula A59 is attached to the end of the sense strand of the siRNA. The end refers to the previous 4 nucleotides from one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula A59 is attached to the end of the sense strand or the antisense strand of the siRNA, and in some embodiments, the P atom in formula A59 is attached to the 3' end of the sense strand of the siRNA. When bound to the above-mentioned position of the sense strand of the siRNA, the siRNA complex shown in formula (308) can release the single siRNA antisense strand when unwound after entering a cell, blocking the process of XO mRNA translating protein and suppressing XO gene expression.

[0335] In some embodiments, the P atom in formula A59 may be bound to any available position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, the P atom in formula A59 may be bound to the 2', 3' or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula A59 is bound to the oxygen atom obtained by dehydrogenating the 3' hydroxyl group of the 3'-terminal nucleotide of the sense strand of the siRNA (in this case, the P atom in A59 can also be regarded as the P atom of the phosphate group contained in the siRNA), or the P atom in formula A59 is bound to the nucleotide by replacing the hydrogen in the 2'-hydroxyl group of one nucleotide in the sense strand of the siRNA, or the P atom in formula A59 is bound to the nucleotide by replacing the hydrogen in the 5' hydroxyl group of the 5'-terminal nucleotide of the sense strand of the siRNA.

[0336] The inventors of the present disclosure have unexpectedly found that the siRNA complex of the present disclosure has significantly improved plasma stability, reduced off-target effects, and also exhibits high XO mRNA silencing activity.In some embodiments, the siRNA of the present disclosure can be one of the siRNAs shown in Tables 1a-1l.The siRNA complexes containing these siRNAs exhibit higher XO mRNA silencing activity.

[0337] [Table 1a]

[0338] [Table 1b]

[0339] [Table 1c]

[0340]

Table 1d

[0341]

Table 1e

[0342]

Table 1f

[0343]

Table 1g

[0344]

Table 1h

[0345]

Table 1i

[0346]

Table 1j

[0347]

Table 1k

[0348]

Table 11

[0349] In the siRNA or siRNA complex described in the present disclosure, adjacent nucleotides are linked by a phosphodiester bond or a thiophosphodiester bond, and the non-bridging oxygen or sulfur atom in the phosphodiester or thiophosphodiester bond may be negatively charged and exist as a hydroxyl group or a sulfhydryl group, and the hydrogen ions in the hydroxyl or sulfhydryl group may be partially or completely replaced by a cation. The cation may be any cation, for example, a metal cation, an ammonium ion NH 4 + In one embodiment, the cation is one or more selected from an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation, in consideration of improving solubility. The alkali metal ion is K + And / or Na +and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and / or an ammonium ion formed by N,N-diisopropylethylamine. Thus, the siRNA or siRNA complex described in the present disclosure may exist at least partially as a salt. In one embodiment, the non-bridging oxygen or sulfur atom in the phosphodiester bond or thiophosphodiester bond is at least partially bound to a sodium ion, and the siRNA or siRNA complex described in the present disclosure exists as a sodium salt or partial sodium salt.

[0350] As those skilled in the art will be aware, modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications.The method of preparing nucleoside monomers with corresponding modifications and the method of introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.All modified nucleoside monomers can be purchased commercially or prepared by known methods.

[0351] <Preparation of siRNA complex represented by formula (308)> The siRNA conjugate of formula (308) may be prepared by any reasonable synthetic route.

[0352] In some embodiments, the siRNA complex shown in formula (308) can be prepared by the following method, which includes sequentially binding nucleoside monomers from 3' to 5' according to the nucleotide types and order of the sense and antisense strands of the siRNA under phosphoramidite solid-phase synthesis conditions, the binding of each nucleoside monomer includes four reactions of deprotection, coupling, capping, oxidation or sulfurization, and isolating and annealing the sense and antisense strands of the siRNA, wherein the siRNA is the siRNA of the present disclosure.

[0353] The method further includes contacting the compound of formula (321) with a nucleoside monomer or a nucleotide sequence bound to a solid support under coupling reaction conditions and in the presence of a coupling reagent to couple the compound of formula (321) to the nucleotide sequence. Hereinafter, the compound of formula (321) is also referred to as a conjugated molecule.

[0354] [ka] During the ceremony, R 4 is a group capable of binding to an siRNA represented by Nu in the compound represented by formula (308). In some embodiments, R 4 is a group capable of binding to the siRNA represented by Nu via a covalent bond. In some embodiments, R 4 is a group that can be conjugated to any functional group of the siRNA represented by Nu via a phosphodiester bond through a reaction, Each S 1 is independently M 1 in which all active hydroxy groups have been replaced with YCOO- groups, and each Y is independently selected from a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, an ethyl group, a n-propyl group, an isopropyl group, a phenyl group, a halophenyl group, and an alkylphenyl group; and in some embodiments, Y is a methyl group.

[0355] n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , M 1 The definitions and selectable ranges of each are as described above.

[0356] R 4is selected to provide for bonding with an N atom on the nitrogen-containing backbone and to provide a suitable reactive site for the synthesis of the siRNA conjugate shown in formula (308). 4 R 2 Linker group or protected R 2 It includes a linker group and a functional group that can be reacted with the siRNA to form the structure shown in A59.

[0357] In some embodiments, R 4 comprises a first functional group capable of forming a phosphite with a group on the siRNA or nucleoside monomer shown as Nu, and a second functional group capable of reacting with a hydroxyl or amino group to form a covalent bond or a solid support bound by said covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxyl or a protected hydroxyl group. In some embodiments, the second functional group is a phosphoramidite, a carboxyl or a carboxylate. In some embodiments, the second functional group is a solid support bound to another part of the molecule via a covalent bond, the covalent bond being formed by a hydroxyl or an amino group. In some embodiments, the solid support is bound via a phosphate ester bond, a carboxyl ester bond or an amide bond. In some embodiments, the solid support is a resin.

[0358] In some embodiments, the first functional group is a hydroxy group, -OR k or a group represented by formula (C3), and the second functional group comprises a structure represented by formula (C1), (C2), (C3), (C1') or (C3').

[0359] [ka] In the formula, q 1 is an integer from 1 to 4, X is O or NH, and M + is a cation and R kis a hydroxy protecting group, SPS represents a solid phase support, JPEG0007610849000047.jpg11126 represents the site where the group is attached to the covalent linking moiety.

[0360] In some embodiments, the first functional group comprises a phosphoramidite group as shown in formula (C3), which can be coupled with a hydroxy group at any position on a nucleotide, for example, a hydroxy group at the 2' position or a hydroxy group at the 3' position, to form a phosphite ester, which can be oxidized or sulfurized to form a phosphodiester bond or a thiophosphate bond as shown in formula A59, and the conjugated molecule can be conjugated to an siRNA. In this case, even if the second functional group is not present, the compound of formula (321) can be conjugated to a nucleotide, and does not affect the acquisition of the siRNA conjugate as shown in formula (308). In this case, after obtaining the sense strand or antisense strand of an siRNA by a method such as phosphoramidite solid-phase synthesis, the compound of formula (321) is reacted with a hydroxy group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a thiophosphate bond is formed in a subsequent oxidation or sulfurization process, and the compound of formula (321) is conjugated to an siRNA.

[0361] In some embodiments, the first functional group comprises a protected hydroxy group. In some embodiments, the second functional group comprises a group capable of reacting with a solid phase support, and the reaction provides a conjugated molecule comprising a solid phase support. In some embodiments, the second functional group comprises a carboxy group, a carboxylate, or a phosphoramidite, as shown in formula (C1), (C2), or (C3). When the second functional group comprises a carboxy group or a carboxylate, the compound of formula (321) is esterified or amidated with a hydroxy group or an amino group on a solid phase support, e.g., a resin, to form a conjugated molecule comprising a solid phase support linked by a carboxylate bond. When the second functional group comprises a phosphoramidite functional group, the compound of formula (321) is coupled with a hydroxy group on a general-purpose solid phase support, e.g., a resin, to form a conjugated molecule comprising a solid phase support linked by a phosphodiester bond, by oxidizing the hydroxy group. Then, starting from the solid support-bound product, nucleoside monomers are sequentially bound according to phosphoramidite solid-phase synthesis to obtain the sense strand or antisense strand of siRNA bound with a conjugated group. In the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with the phosphoramidite group in the nucleoside monomer under coupling reaction conditions.

[0362] In some embodiments, the first functional group comprises a hydroxy group or a protected hydroxy group, and the second functional group comprises a solid-phase support bound by a carboxylic acid ester bond, or a solid-phase support bound by an amide bond, or a solid-phase support bound by a phosphate ester bond, as shown in formula (C1') or (C3').In this case, use the compound of formula (321) as starting material instead of a solid-phase support, and sequentially bind nucleoside monomers according to phosphoramidite solid-phase synthesis method to obtain the sense strand or antisense strand of siRNA bound with a conjugated group.

[0363] In some embodiments, the carboxylate is -COO - M + where M +is a cation, e.g., a metal cation, the ammonium cation NH 4 + In one embodiment, the metal ion is an alkali metal ion, such as K + Or Na + In consideration of improving solubility and smooth reaction, in some embodiments, the organic ammonium ion is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation, for example, an ammonium ion formed by triethylamine or an ammonium ion formed by N,N-diisopropylethylamine. In some embodiments, the carboxylate is a triethylamine carboxylate or an N,N-diisopropylethylamine carboxylate.

[0364] In some embodiments, R 4 includes a structure represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11') or (B12').

[0365] [ka] In the formula, q 1 is an integer from 1 to 4, and q 2 is an integer from 1 to 10, X is O or NH, and M + is a cation and R k is a hydroxy protecting group, SPS represents a solid phase support, JPEG0007610849000049.jpg10140 represents the site at which the group is attached to the covalent linking moiety. 1 is 1 or 2. In some embodiments, q 2 is an integer from 1 to 5. In some embodiments, R 4 In some embodiments, R 4 includes a structure represented by formula (B11) or (B12).

[0366] In some embodiments, R k is one or more of Tr (trityl group), MMTr (4-methoxytrityl group), DMTr (4,4'-bismethoxytrityl group), TMTr (4,4',4''-trimethoxytrityl group). In some embodiments, R k may be DMTr, ie, 4,4'-dimethoxytrityl.

[0367] L 1 The definition is as described above.

[0368] In some embodiments, L 1 is M 1 A targeting group is attached to the N atom on the nitrogen-containing backbone and is used to provide liver targeting functionality to the siRNA complex shown in formula (308). In some embodiments, L 1 includes any one of A1 to A26 or a combination thereof.

[0369] From the above description, as can be easily understood by those skilled in the art, compared to the phosphoramidite solid phase synthesis method known in the art, the above first functional group and any second functional group can provide the siRNA complex shown in formula (308), in which the conjugate molecule is bound to any possible position of the nucleotide sequence, for example, the end of the nucleotide sequence, the terminus of the nucleotide sequence. Accordingly, unless otherwise specified, in the following description of the preparation of the siRNA complex and / or conjugate molecule, when reactions such as "deprotection", "coupling", "capping", "oxidation", "sulfurization" are mentioned, it should be understood that the reaction conditions and reagents related to the phosphoramidite nucleic acid solid phase synthesis method known in the art also apply to these reactions. Exemplary reaction conditions and reagents are described in detail below.

[0370] In some embodiments, each S 1 is independently M 1 In some embodiments, each S1 is independently M 1 In some embodiments, at least one active hydroxy group is protected with a hydroxy protecting group. 1 is independently M 1 In some embodiments, any hydroxy protecting group known to those skilled in the art may be used in the present invention. 1 In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, where each Y is independently C 1 -C 10 Alkyl group and C 6 -C 10 aryl groups, 1 -C 10 Alkyl group and C 6 -C 10 The aryl group is optionally substituted with one or more substituents, the substituents being halogen and C 1 In some embodiments, each Y is independently selected from the group consisting of a methyl group, a trifluoromethyl group, a difluoromethyl group, a monofluoromethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, an ethyl group, a n-propyl group, a isopropyl group, a phenyl group, a halophenyl group, and a C 1 -C 6 alkylphenyl groups.

[0371] In some embodiments, each S 1 are each independently selected from the group consisting of formulae A46 to A54.

[0372] [ka]

[0373] In some embodiments, S 1 is formula A49 or A50.

[0374] In some embodiments, each Y is independently selected from a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, an ethyl group, a n-propyl group, a isopropyl group, a phenyl group, a halophenyl group, and an alkylphenyl group, and in some embodiments, Y is a methyl group.

[0375] As described above, the method for preparing the siRNA complex shown in formula (308) further includes the steps of synthesizing the other strand of the siRNA (for example, if the sense strand of the siRNA bound to the complex molecule is synthesized in the above step, the method further includes synthesizing the antisense strand of the siRNA according to a solid-phase synthesis method, and vice versa), isolating the sense strand and the antisense strand, and annealing them. Specifically, in the isolation step, the solid-phase support bound to the nucleotide sequence and / or the complex molecule is cleaved and necessary protecting groups are removed (in this case, each S in the compound of formula (321) is cleaved). 1 The group is the corresponding M 1 The siRNA complex shown in formula (308) is obtained by annealing the sense strand (or antisense strand) of the siRNA to which the conjugate molecule is bound, and the corresponding antisense strand (or sense strand) of the siRNA to which the conjugate molecule is bound.

[0376] In some embodiments, the method for preparing the siRNA complex represented by formula (308) comprises contacting a compound represented by formula (321) with a first nucleoside monomer at the 3' end of a sense strand or an antisense strand under coupling reaction conditions and in the presence of a coupling reagent, thereby coupling the compound represented by formula (321) to the first nucleotide in the sequence, and synthesizing the sense strand or antisense strand of the siRNA by sequentially coupling the nucleoside monomers from 3' to 5' according to the type and order of nucleotides of the desired sense strand or antisense strand under phosphoramidite solid phase synthesis conditions, wherein the compound represented by formula (321) is R 4a step of deprotecting the compound of formula (321) before binding with a first nucleoside monomer, and the binding of each nucleoside monomer includes four reactions, namely, deprotection, coupling, capping, oxidation, or sulfurization, to obtain a sense strand or antisense strand of a nucleic acid to which a composite group is bound; a step of sequentially binding nucleoside monomers from 3' to 5' under phosphoramidite solid-phase synthesis conditions according to the type and order of nucleotides in the antisense strand or sense strand to synthesize an antisense strand or sense strand of a nucleic acid, and the binding of each nucleoside monomer includes four reactions, namely, deprotection, coupling, capping, oxidation, or sulfurization, to remove the protecting groups, cleaving from the solid-phase support, isolating and purifying to obtain a sense strand and an antisense strand, and annealing the resulting mixture.

[0377] In some embodiments, the method for preparing the siRNA complex represented by formula (308) includes the steps of: synthesizing a sense strand and an antisense strand by sequentially coupling nucleoside monomers from 3' to 5' according to the type and order of nucleotides of the sense strand or the antisense strand in the double-stranded siRNA, and the coupling of each nucleoside monomer includes four reactions, namely, deprotection, coupling, capping, oxidation, or sulfurization, to obtain a sense strand bound to a solid support and an antisense strand bound to a solid support; contacting a compound represented by formula (321) with the sense strand bound to a solid support or the antisense strand bound to a solid support under coupling reaction conditions and in the presence of a coupling reagent; 4 The method includes the steps of binding a compound of formula (321) containing a first functional group which is a phosphoramidite group to a sense strand or an antisense strand, removing the protecting group and cleaving the compound from the solid support, isolating and purifying the compound, respectively, to obtain a sense strand or an antisense strand of siRNA bound to a conjugated group, and annealing the compound.

[0378] In some embodiments, the P atom in formula A59 is bound to the 3' end of the sense strand in the siRNA, and the method for preparing the siRNA complex represented by formula (308) includes the steps of: (1) Compound of formula (321) (Compound of formula (321) is R 4 , a protected hydroxy group OR k and a second functional group having a structure represented by formula (C1') or (C3'). k and contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer attached to the solid support by a conjugate molecule. (2) synthesizing the sense strand of siRNA in the 3'-5' direction by phosphoramidite solid-phase synthesis starting from the nucleoside monomer bound to the solid support by the conjugate molecule; (3) synthesizing the antisense strand of the siRNA by phosphoramidite solid phase synthesis; (4) isolating and annealing the sense and antisense strands of the siRNA to obtain a siRNA complex represented by formula (308).

[0379] In step (1), the protecting group R in the compound of the above formula (321) k The method of removing includes contacting the compound of Formula (321) with a deprotection reagent under deprotection conditions. The deprotection conditions include a temperature of 0 to 50° C., in some embodiments 15 to 35° C., a reaction time of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, in some embodiments dichloroacetic acid. The molar ratio of the deprotection reagent to the compound of Formula (321) is 10:1 to 1000:1, in some embodiments 50:1 to 500:1.

[0380] The coupling reaction conditions and coupling reagents may be any conditions and reagents suitable for the coupling reaction, and in some embodiments may be the same as those for the coupling reaction in the solid phase synthesis method employed.

[0381] In some embodiments, the coupling reaction conditions include a reaction temperature of 0 to 50° C., and in some embodiments, 15 to 35° C. The molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1 to 1:50, and in some embodiments, 1:2 to 1:5. The molar ratio of the compound of formula (321) to the coupling reagent may be 1:1 to 1:50, and in some embodiments, 1:3 to 1:10, and the reaction time is 200 to 3000 seconds, and in some embodiments, 500 to 1500 seconds. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, and in some embodiments, 5-ethylthio 1H-tetrazole. The coupling reaction may be carried out in an organic solvent, and the organic solvent is one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile. For the compound of formula (321), the dosage of the organic solvent is 3-50 L / mol, in some embodiments 5-20 L / mol.

[0382] In step (2), starting from the nucleoside monomer bound to the solid support by the conjugated molecule prepared in the above step, synthesize the sense strand SS of the second siRNA conjugate in the 3'-5' direction by phosphoramidite nucleic acid solid phase synthesis method, in which the conjugated group is bound to the 3' end of the obtained sense strand.

[0383] Other conditions for the solid phase synthesis in steps (2) and (3) include deprotection conditions for nucleoside monomers, the type and dosage of deprotection reagent, coupling reaction conditions, the type and dosage of coupling reagent, capping reaction conditions, the type and dosage of capping reagent, oxidation reaction conditions, the type and dosage of oxidation reagent, sulfurization reaction conditions, and the type and dosage of sulfurization reagent, and various reagents, dosages and conditions commonly used in this field are adopted.

[0384] For example, in some embodiments, in steps (2) and (3), the solid phase synthesis may use the following conditions:

[0385] The deprotection conditions for the nucleoside monomer are as follows: temperature is 0 to 50°C, in some embodiments 15 to 35°C, reaction time is 30 to 300 seconds, in some embodiments 50 to 150 seconds, the deprotection reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, in some embodiments dichloroacetic acid, and the molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support may be 2:1 to 100:1, in some embodiments 3:1 to 50:1.

[0386] The coupling reaction conditions are as follows: temperature is 0 to 50°C, and in some embodiments 15 to 35°C; the molar ratio of the nucleic acid sequence bound to the solid phase carrier to the nucleoside monomer may be 1:1 to 1:50, and in some embodiments 1:5 to 1:15; the molar ratio of the nucleic acid sequence bound to the solid phase carrier to the coupling reagent is 1:1 to 1:100, and in some embodiments 1:50 to 1:80; and the reaction time and selection of the coupling reagent are the same as above.

[0387] The capping reaction conditions are as follows: temperature is 0 to 50°C, in some embodiments 15 to 35°C, reaction time is 5 to 500 seconds, in some embodiments 10 to 100 seconds, and the selection of the capping reagent is the same as above. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence bound to the solid phase carrier is 1:100 to 100:1, in some embodiments 1:10 to 10:1. When equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid phase carrier may be 1:1:10 to 10:10:1, in some embodiments 1:1:2 to 2:2:1.

[0388] The oxidation reaction conditions are as follows: temperature is 0 to 50°C, in some embodiments 15 to 35°C; reaction time is 1 to 100 seconds, in some embodiments 5 to 50 seconds; and the oxidation reagent is iodine in some embodiments (provided as iodine water in some embodiments). The molar ratio of the oxidation reagent to the nucleic acid sequence bound to the solid phase support in the coupling step may be 1:1 to 100:1, in some embodiments 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The sulfurization reaction conditions are as follows: temperature is 0 to 50°C, in some embodiments 15 to 35°C; reaction time is 50 to 2000 seconds, in some embodiments 100 to 1000 seconds; and the sulfurization reagent is xanthan hydride in some embodiments. The molar ratio of the sulfurization reagent to the nucleic acid sequence bound to the solid phase carrier in the coupling step is 10:1 to 1000:1, and in some embodiments, 10:1 to 500:1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:3 to 3:1.

[0389] After all nucleoside monomers are linked and before annealing, the method further includes isolating the sense and antisense strands of the siRNA. Isolation methods are known to those skilled in the art and generally include cleaving the synthesized nucleotide sequence from the solid support, removing the protecting groups on the bases, phosphate groups and ligands, purifying and desalting.

[0390] The synthesized nucleotide sequence may be cleaved from the solid support and the protecting groups on the bases, phosphate groups, and ligands may be removed by the usual cleavage and deprotection methods used in siRNA synthesis. For example, the obtained nucleotide sequence bound to the solid support is contacted with concentrated aqueous ammonia, and in the deprotection process, the protecting groups YCOO- of A46 to A54 are converted to hydroxy groups, and S 1 The group is replaced with the corresponding M 1group to produce a siRNA complex represented by formula (308). Here, the concentrated ammonia water may be 25 to 30% by weight of ammonia water, and the amount of concentrated ammonia water may be 0.2 ml / μmol to 0.8 ml / μmol with respect to the target siRNA sequence.

[0391] If the synthesized nucleotide sequence has at least one 2'-TBDMS protection, the method further comprises removing the 2'-TBDMS protection by contacting the nucleotide sequence from which the solid support has been removed with triethylamine trihydrofluoride. In this case, the corresponding nucleotide in the obtained target siRNA sequence has a free 2'-hydroxy group. The dosage of pure triethylamine trihydrofluoride may be 0.4ml / μmol to 1.0ml / μmol relative to the target siRNA sequence. In this way, the siRNA complex shown in formula (308) can be obtained.

[0392] Methods of purification and desalting are well known to those skilled in the art. For example, purification of nucleic acids can be completed using a preparative ion chromatography purification column with gradient elution of NaBr or NaCl, and the products can be collected and combined before being desalted using a reverse phase chromatography purification column.

[0393] In the siRNA complex thus obtained, the non-bridging oxygen or sulfur atom in the internucleotide phosphodiester or thiophosphodiester bond is essentially bound to a sodium ion, and the siRNA complex of formula (308) is essentially present as a sodium salt. The sodium ion can be replaced with hydrogen ions and / or other cations by well-known ion exchange methods to obtain other forms of the siRNA complex of formula (308). The cations are as described above.

[0394] In the synthesis process, the purity and molecular weight of the nucleic acid sequence can be constantly detected to better control the synthesis quality. Such detection methods are known to those skilled in the art. For example, the purity of the nucleic acid can be detected by ion exchange chromatography, and the molecular weight can be measured by liquid chromatography tandem mass spectrometry (LC-MS).

[0395] The annealing method is also well known to those skilled in the art. For example, a sense strand (S strand) and an antisense strand (AS strand) that are simply synthesized can be mixed in an equimolar ratio with water for injection, heated to 70-95°C, and then cooled at room temperature to form a double-stranded structure by hydrogen bonding. In this way, the siRNA complex shown in formula (308) can be obtained.

[0396] After obtaining the siRNA complex, in some embodiments, the synthesized siRNA complex represented by formula (308) can be characterized by molecular weight detection or the like using a method such as liquid chromatography tandem mass spectrometry, and it can be confirmed that the synthesized siRNA complex is the siRNA complex represented by formula (308) of the intended design and that the sequence of the synthesized siRNA is the desired siRNA sequence, for example, one of the sequences shown in Table 1.

[0397] The compound of formula (321) can be prepared by a process which comprises contacting a compound of formula (313) with a cyclic acid anhydride in an organic solvent under esterification conditions and in the presence of a base and an esterification catalyst, followed by ion exchange and isolation to obtain a compound of formula (321).

[0398] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1、 S 1The definitions and selectable ranges of each are as described above. R 6 is R in Eq. (321). 4 In some embodiments, R 6 has the structure shown in formula (A61).

[0399] [ka] In the formula, R i can achieve bonding with the N atom on the nitrogen-containing backbone, R k Any group having one free hydroxyl group bonded to O, R k is a hydroxy protecting group. In this case, R 4 contains a first functional group as a hydroxy-protecting group and a second functional group, and the second functional group contains a structure represented by formula (C1) or (C2), thereby obtaining a compound of formula (321).

[0400] The esterification reaction conditions are a reaction temperature of 0 to 100° C. and a reaction time of 8 to 48 hours, and in some embodiments, the esterification reaction conditions are a reaction temperature of 10 to 40° C. and a reaction time of 20 to 30 hours.

[0401] In some embodiments, the organic solvent includes one or more of an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ethyl ether and / or methyl tert-butyl ether, and the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The dosage of the organic solvent is 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol, relative to the compound represented by formula (313).

[0402] In some embodiments, the cyclic acid anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments, succinic anhydride. The molar ratio of the cyclic acid anhydride to the compound represented by formula (313) is 1:1 to 10:1, and in some embodiments, 2:1 to 5:1.

[0403] The esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound represented by formula (313) is 1:1 to 10:1, and in some embodiments, is 2:1 to 5:1.

[0404] In some embodiments, the base may be any inorganic base, organic base, or a combination thereof. In consideration of solubility and product stability, the base may be, for example, a tertiary amine. In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by formula (313) is 1:1 to 20:1, and in some embodiments, is 3:1 to 10:1.

[0405] The ion exchange action is to convert the compound of formula (321) into the desired carboxylic acid or carboxylate form, and the ion exchange method is known to those skilled in the art, and can be carried out by using appropriate ion exchange solutions and exchange conditions, such as M + A complex molecule having a cation can be obtained, and detailed description is omitted here. In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is 0.2-0.8 M, in some embodiments, the concentration of the triethylamine phosphate solution is 0.4-0.6 M, and the dosage of the triethylamine phosphate solution is 3-6 L / mol, and in further embodiments, 4-5 L / mol, relative to the compound of formula (313).

[0406] The compound of formula (321) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the compound of formula (321) can be isolated by a chromatography method, for example, by two chromatographic conditions: (1) normal phase purification silica gel: 200-300 mesh silica gel packing material is gradient eluted with dichloromethane:methanol=100:18-100:20 containing 1wt‰ triethylamine, or (2) reverse phase purification: C18, C8 reverse phase packing material is gradient eluted with methanol:acetonitrile=0.1:1-1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be used directly in the subsequent reaction.

[0407] In some embodiments, the method for preparing a compound of formula (321) further comprises contacting the product obtained by the ion exchange reaction with a solid support further comprising an amino group or a hydroxy group in the presence of a condensation agent, a condensation catalyst, and a tertiary amine in an organic solvent under condensation reaction conditions. 4 contains a first functional group and a second functional group, the first functional group contains a hydroxy protecting group, and the second functional group contains the structure shown in formula (C1'), to obtain a compound of formula (321).

[0408] The solid phase carrier is one of the carriers used in solid phase synthesis of siRNA, some of which are known to those skilled in the art. For example, the solid phase carrier may be selected from solid phase carriers containing active hydroxyl or amino functional groups, and in some embodiments, the solid phase carrier is an amino resin or a hydroxy resin. In some embodiments, the amino resin or the hydroxy resin has parameters of a particle size of 100 to 400 mesh and a surface loading of amino or hydroxyl groups of 0.2 to 0.5 mmol / g. The dosage ratio of the compound represented by formula (321) to the solid phase carrier is 10 to 400 μmol compound / gram of solid phase carrier (μmol / g). In some embodiments, the dosage ratio of the compound represented by formula (321) to the solid phase carrier is 50 to 200 μmol / g.

[0409] The organic solvent may be any suitable solvent or mixture of solvents known to those skilled in the art. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvent, ether solvent, halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ethyl ether and / or methyl tert-butyl ether, and the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. For the compound of formula (321), the dosage of the organic solvent is 20-200 L / mol, and in some embodiments, 50-100 L / mol.

[0410] In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate (PyBop), 3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) and / or O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate), and in some embodiments, the condensing agent is O-benzotriazol-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (321) is 1:1 to 20:1, and in other embodiments, is 1:1 to 5:1.

[0411] In some embodiments, the tertiary amine is triethylamine and / or N,N-diisopropylethylamine, and in some embodiments, N,N-diisopropylethylamine, and the molar ratio of the tertiary amine to the compound represented by Formula (321) is 1:1 to 20:1, and in some embodiments, 1:1 to 5:1.

[0412] In some embodiments, the method for preparing the compound of formula (321) may further include contacting the resulting condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions, and isolating to obtain the compound represented by formula (321). The function of the capping reaction is to remove any active reactive functional groups that have not yet reacted completely, to avoid the generation of unwanted by-products in subsequent reactions. The conditions of the capping reaction are a reaction temperature of 0 to 50°C, in some embodiments 15 to 35°C, and a reaction time of 1 to 10 h, in some embodiments 3 to 6 h. The capping reagent may be a capping reagent used in siRNA solid-phase synthesis, known to those skilled in the art.

[0413] In some embodiments, the capping reagent comprises capping reagent 1 (cap1) and capping reagent 2 (cap2), where capping reagent 1 is N-methylimidazole, and in some embodiments, N-methylimidazole is provided as a pyridine / acetonitrile mixed solution, where the volume ratio of pyridine to acetonitrile is 1:10 to 1:1, and in some embodiments, 1:3 to 1:1, and the volume ratio of the total volume of pyridine and acetonitrile to N-methylimidazole is 1:1 to 10:1, and in some embodiments, 3:1 to 7:1. The capping reagent 2 is acetic anhydride. In some embodiments, the capping reagent 2 is provided as an acetonitrile solution of acetic anhydride, where the volume ratio of acetic anhydride to acetonitrile is 1:1 to 1:10, and in further embodiments, 1:2 to 1:6.

[0414] In some embodiments, the ratio of the volume of the pyridine / acetonitrile solution of N-methylimidazole to the mass of the compound of formula (321) is 5 ml / g to 50 ml / g, and in some embodiments, 15 ml / g to 30 ml / g. The ratio of the volume of the acetonitrile solution of acetic anhydride to the mass of the compound of formula (321) is 0.5 ml / g to 10 ml / g, and in some embodiments, 1 ml / g to 5 ml / g.

[0415] In some embodiments, equimolar amounts of acetic anhydride and N-methylimidazole are used as capping reagents. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethylsulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The dosage of the organic solvent relative to the compound of formula (321) is 10 to 50 L / mol, and in some embodiments, 5 to 30 L / mol.

[0416] In some embodiments, the acylation catalyst may be selected from any catalyst that can be used in esterification condensation or amidation condensation, such as an alkali heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1 to 1:1, and in some embodiments, is 0.01:1 to 0.1:1.

[0417] In some embodiments, the compound of formula (321) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, the compound of formula (321) can be obtained by thorough washing with an organic solvent and filtration to remove unreacted reactants, excess capping reagent, and other impurities. The organic solvent is selected from acetonitrile, dichloromethane, methanol, and in some embodiments, acetonitrile.

[0418] In some embodiments, a method for preparing a conjugated molecule of formula (321) comprises contacting a compound of formula (313) with a phosphorodiamidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolating to obtain a compound of formula (321). 4contains a first functional group and a second functional group, the first functional group contains a hydroxy protecting group, and the second functional group contains a structure shown in formula (C3), to obtain a compound of formula (321).

[0419] In some embodiments, the coupling reaction conditions may be a temperature of 0 to 50°C, for example 15 to 35°C. The molar ratio of the compound of formula (313) to the phosphorodiamidite may be 1:1 to 1:50, for example 1:5 to 1:15, and the molar ratio of the compound of formula (313) to the coupling reagent may be 1:1 to 1:100, for example 1:50 to 1:80. The reaction time may be 200 to 3000 seconds, for example 500 to 1500 seconds. The phosphorodiamidite may be, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, may be commercially available, or may be synthesized by a method known in the art. The coupling reagent may be one or more selected from 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, for example 5-ethylthio 1H-tetrazole. The coupling reaction may be carried out in an organic solvent, and the organic solvent may be one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, for example, anhydrous acetonitrile. In some embodiments, the amount of the organic solvent may be 3 to 50 L / mol, for example, 5 to 20 L / mol, relative to the compound of formula (313). By carrying out the coupling reaction, a hydroxy group in the compound of formula (313) is reacted with a phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be used in the subsequent reaction as it is.

[0420] In some embodiments, the method for preparing the compound of formula (321) further comprises contacting the isolated product with a hydroxyl-containing solid support under coupling reaction conditions in an organic solvent in the presence of a coupling reagent, followed by capping, oxidation, and isolation to obtain the compound of formula (321). In this case, R 4 contains a first functional group and a second functional group, the first functional group contains a hydroxy protecting group, and the second functional group has the structure shown in formula (C3'), to obtain a compound of formula (321).

[0421] In some embodiments, the solid support is a solid support that can be used for solid-phase synthesis of nucleic acids known in the art, for example, a commercially available, general-purpose solid support that has been deprotected (NittoPhase® HL UnyLinker TM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, the structure of which is shown in formula B80).

[0422] [ka]

[0423] The deprotection reaction is known to those skilled in the art. In some embodiments, the deprotection conditions are a temperature of 0 to 50°C, for example 15 to 35°C, and a reaction time of 30 to 300 seconds, for example 50 to 150 seconds. The deprotection reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group in the stationary phase is 2:1 to 100:1, for example 3:1 to 50:1. By carrying out the deprotection, a free hydroxyl group having a reaction activity is obtained on the surface of the solid phase support, which makes it easier to carry out the next coupling reaction.

[0424] The coupling reaction conditions and the selection of the coupling reagent are as described above. By carrying out the coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite bond.

[0425] In some embodiments, the capping reaction conditions are a temperature of 0 to 50° C., for example, 15 to 35° C., a reaction time of 5 to 500 seconds, for example, 10 to 100 seconds, and the capping reaction is carried out in the presence of a capping reagent. The selection and dosage of the capping reagent are as described above.

[0426] The oxidation reaction conditions may be a temperature of 0 to 50°C, for example 15 to 35°C, a reaction time of 1 to 100 seconds, for example 5 to 50 seconds, and the oxidation reagent may be, for example, iodine (provided as iodine water in some embodiments). In some embodiments, the molar ratio of the oxidation reagent to the nucleic acid sequence bound to the solid phase carrier may be 1:1 to 100:1, for example 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1 to 1:1:3.

[0427] In some embodiments, R 6 is one of the groups of formula B7 or B8.

[0428] [ka] In the formula, q 2 The definition is as described above.

[0429] In this case, the compound represented by formula (313) can be obtained by a preparation method in which the compound represented by formula (314) is contacted with the compound represented by formula (A-1) or the compound represented by formula (A-2) in an organic solvent under amidation reaction conditions and in the presence of an amidation reaction condensing agent and a tertiary amine, and then isolated.

[0430] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1 , S 1 , q 2 and R k The definitions and selectable ranges of each are as described above.

[0431] The amidation reaction conditions may be a reaction temperature of 0 to 100° C. and a reaction time of 1 to 48 hours, and in some embodiments, the amidation reaction conditions are a reaction temperature of 10 to 40° C. and a reaction time of 2 to 16 hours.

[0432] In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the alcohol solvent is one or more of methanol, ethanol, and propanol, and in some embodiments, is ethanol. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent is 3 to 50 L / mol relative to the compound of formula (314), and 3 to 20 L / mol in further embodiments.

[0433] In some embodiments, the amidation reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate / ester, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazole-tetramethyluronium hexafluorophosphate / ester, and in further embodiments, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one. The molar ratio of the amidation reaction condensing agent to the compound represented by formula (314) may be 1:1 to 10:1, and in some embodiments, is 2.5:1 to 5:1.

[0434] In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine, and in further embodiments, N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound represented by Formula (314) is 3:1 to 20:1, and in some embodiments, 5:1 to 10:1.

[0435] In some embodiments, compounds of formula (A-1) and formula (A-2) may be prepared by any suitable method. For example, R k is a DMTr group, compounds of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl. Similarly, compounds of formula (A-2) can be prepared by contacting 3-amino-1,2-propanediol with a cyclic acid anhydride, which may have 4 to 13 carbon atoms, and in some embodiments 4 to 8 carbon atoms, with the cyclic acid anhydride being one of these. As can be readily appreciated by those skilled in the art, the choice of the cyclic acid anhydride depends on the q in compounds of formula (A-2). 2For example, when the cyclic anhydride is succinic anhydride, q 2 = 1 and the cyclic acid anhydride is glutaric anhydride, q 2 =2, which can be inferred.

[0436] In some modifications, the compound of formula (313) can be prepared by reacting the compound of formula (314) with the cyclic acid anhydride, 3-amino-1,2-propanediol, and DMTrCl in sequence. As can be easily understood by those skilled in the art, these modifications do not affect the structure and function of the compound of formula (313), and can be easily realized by those skilled in the art by the above method.

[0437] Similarly, the compound of formula (313) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is removed by evaporation, the compound of formula (313) can be isolated by a chromatography method, for example, by two chromatography conditions: (1) normal phase purification silica gel: 200-300 mesh silica gel packing is gradient eluted with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1: 1: 1: 0.5-1: 1: 1: 0.6, and (2) reverse phase purification: C18, C8 reverse phase packing is gradient eluted with methanol: acetonitrile = 0.1: 1-1: 0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be used directly in the subsequent reaction.

[0438] In some embodiments, the compound of formula (314) can be obtained by a preparation process comprising contacting a compound of formula (320) with a compound of formula (316) in the presence of an amidation reaction condensing agent and a tertiary amine under condensation reaction conditions in an organic solvent, followed by isolation.

[0439] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 The definitions and selectable ranges of each are as described above.

[0440] The compound of formula (316) may be, for example, a compound disclosed in J. Am. Chem. Soc. 2014, 136, 16958-16961. Alternatively, the compound of formula (316) can be prepared by a person skilled in the art by various methods. For example, some compounds of formula (316) can be prepared by referring to the method disclosed in Example 1 of U.S. Patent US 8,106,022 B2, the entire contents of which are incorporated herein by reference.

[0441] In some embodiments, the condensation reaction conditions are a reaction temperature of 0 to 100° C. and a reaction time of 0.1 to 24 hours, and in some embodiments, a reaction temperature of 10 to 40° C. and a reaction time of 0.5 to 16 hours.

[0442] Considering the structure of the compound of formula (314), which is the desired product, the molar ratio of the compound of formula (316) to the compound of formula (320) should be determined based on the sum of n1 and n3 in formula (320). In some embodiments, for example, when n1+n3=3, in order to ensure that the reaction is complete and not excessive, the molar ratio of the compound of formula (316) to the compound of formula (320) may be 3:1 to 3.5:1, and in some embodiments, is 3.01:1 to 3.15:1.

[0443] In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvent, ether solvent, halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ethyl ether and / or methyl tert-butyl ether, the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane, and the organic solvent is dichloromethane. For the compound of formula (320), the dosage of the organic solvent is 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol.

[0444] In some embodiments, the amidation reaction condensing agent is one or more of benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate / ester, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT), O-benzotriazole-tetramethyluronium hexafluorophosphate / ester, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, or 1-hydroxybenzotriazole, and in further embodiments, a mixture of benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate / ester and 1-hydroxybenzotriazole, where benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate / ester and 1-hydroxybenzotriazole are used in equimolar amounts. The molar ratio of the total amidation reaction condensing agent to the compound represented by formula (316) may be 1:1 to 3:1, and in some embodiments, is 1.05:1 to 1.5:1.

[0445] The tertiary amine can be N-methylmorpholine, triethylamine, or N,N-diisopropylethylamine, and in some embodiments is N-methylmorpholine, and the molar ratio of the tertiary amine to the compound represented by Formula (316) can be from 2:1 to 10:1, and in some embodiments, is from 2:1 to 5:1.

[0446] Similarly, the compound of formula (314) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the compound of formula (314) can be isolated by a chromatography method, for example, by two chromatography conditions: (1) normal phase purification silica gel: 200-300 mesh silica gel packing material, gradient elution with dichloromethane:methanol=100:5-100:7, and (2) reverse phase purification: C18, C8 reverse phase packing material, gradient elution with methanol:acetonitrile=0.1:1-1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be used directly in the subsequent reaction.

[0447] Compounds of formula (320) can be obtained commercially or by known methods by one of ordinary skill in the art. For example, m1=m2=m3=3, n1=1, n3=2, and each R 10 , R 11 , R 12 , R 13 , R 14 , R 15 When both are H, the compound of formula (320) can be purchased commercially from Alfa Acer.

[0448] The siRNA complex of the present disclosure may be used in combination with other pharma- ceutically acceptable additives, which may be one or more of various preparations or compounds commonly used in the art; for details, see the description of the pharmaceutical composition of the present disclosure above.

[0449] Use of the siRNA, drug composition, and siRNA complex of the present disclosure In some embodiments, the present disclosure provides a use of the siRNA, and / or drug composition and / or siRNA complex of the present disclosure in the preparation of a drug for the treatment and / or prevention of uric acid metabolism disorder or a disease or physiological condition caused by uric acid metabolism disorder. In some embodiments, the disease or physiological condition caused by uric acid metabolism disorder is hyperuricemia or gout.

[0450] In some embodiments, the present disclosure provides a method for preventing and / or treating uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders, comprising administering an effective amount of the siRNA and / or drug composition and / or siRNA complex of the present disclosure to a subject in need thereof. In some embodiments, the disease or physiological condition caused by uric acid metabolism disorders is hyperuricemia or gout.

[0451] By administering the siRNA active ingredient of the present disclosure to a subject in need thereof, the purpose of preventing and / or treating uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders can be achieved by RNA interference mechanism. Therefore, the siRNA and / or drug composition and / or siRNA complex of the present disclosure can be used for preventing and / or treating uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders, or can be used for preparing drugs for preventing and / or treating uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders. In some embodiments, the uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders are hyperuricemia or gout.

[0452] The term "pharmaceutical administration / administration" as used herein refers to the introduction of the siRNA, drug composition and / or siRNA complex of the present disclosure into the body of a subject by a method or route that allows at least a portion of the siRNA, drug composition and / or siRNA complex of the present disclosure to be localized at a desired site to produce a desired effect. Suitable administration routes for the method of the present disclosure include local administration and systemic administration. Generally, local administration delivers more siRNA complex to a specific site than to the subject's systemic circulation, while systemic administration delivers the siRNA, drug composition and / or siRNA complex of the present disclosure to the subject's basic systemic circulation. Considering that the present disclosure is intended to provide a means for preventing and / or treating uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders, in some embodiments, an administration method that can deliver the drug to the liver is adopted.

[0453] Administration to a subject may be by any suitable route known in the art, including, but not limited to, oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, intratracheal (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. The frequency of administration may be once or more per day, week, biweekly, triweekly, monthly, or yearly.

[0454] The dose of the siRNA, drug composition or siRNA complex described in this disclosure may be a dose that is conventional in the art, and the dose may be determined according to various parameters, particularly the age, weight and sex of the subject. Toxicity and therapeutic efficacy are measured in cell cultures or experimental animals by standard pharmaceutical procedures, for example, LD 50 (the dose that kills 50% of the colony) and ED 50 (For a quantitative response, this refers to the dose capable of eliciting 50% of the maximum response strength; for a qualitative response, this refers to the dose at which a positive response occurs in 50% of experimental subjects) may be measured. A range of human dosages can be derived based on data obtained from cell culture assays and animal studies.

[0455] When the siRNA, drug composition, and / or siRNA complex described in the present disclosure is administered to, for example, male or female, 6-12 week old, C57BL / 6J mouse weighing 18-25 g or ob / ob mouse weighing 30-45 g, the amount of siRNA is as follows: (i) for the siRNA complex, the siRNA dose may be 0.001-100 mg / kg body weight, in some embodiments, 0.01-50 mg / kg body weight, in some embodiments, 0.05-20 mg / kg body weight, in some embodiments, 0.1-15 mg / kg body weight, and in some embodiments, 0.1-10 mg / kg body weight; and (ii) for the drug composition formed from the siRNA and a pharma- ceutically acceptable carrier, the siRNA dose may be 0.001-50 mg / kg body weight, in some embodiments, 0.01-10 mg / kg body weight, in some embodiments, 0.05-5 mg / kg body weight, and in some embodiments, 0.1-3 mg / kg body weight.

[0456] In some embodiments, the present disclosure provides a method for suppressing expression of XO gene in a cell, comprising contacting an effective amount of siRNA and / or drug composition and / or siRNA complex of the present disclosure with the cell, and introducing the siRNA and / or drug composition and / or siRNA complex of the present disclosure into the cell, thereby achieving the purpose of suppressing expression of XO gene in the cell by RNA interference mechanism. The cell may be selected from cancer cell lines such as SMMC-7721, CAL-27, Huh7, etc., or isolated primary hepatocytes. In some embodiments, the cell is a CAL-27 cell.

[0457] The method provided by the present disclosure silences expression of the XO gene in cells. The siRNA dosage in the provided modified siRNA, drug composition and / or siRNA complex is generally an amount capable of reducing expression of the target gene and resulting in an extracellular concentration at the surface of the target cell of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to about 5 nM. The amount required to achieve this local concentration will vary depending on a variety of factors, including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cell or tissue, the delivery route (local or systemic), etc. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

[0458] <Kit> The present disclosure provides kits comprising an effective amount of at least one of the modified siRNAs, drug compositions, and siRNA complexes of the present disclosure.

[0459] In some embodiments, the kits described herein may provide the modified siRNA in a container. In some embodiments, the kits described herein may include a container providing a pharma- ceutically acceptable excipient. In some embodiments, the kits may include other components, such as stabilizers or preservatives. In some embodiments, the kits described herein may include at least one other therapeutic agent in a container separate from the container providing the modified siRNA described herein. In some embodiments, the kits may include instructions for mixing the modified siRNA with a pharma- ceutical acceptable carrier and / or additives or other components, if present.

[0460] In the kit of the present disclosure, the modified siRNA and pharma- ceutically acceptable carrier and / or additive, and the modified siRNA, drug composition and / or siRNA complex, and / or pharma- ceutically acceptable additive may be provided in any form, for example, liquid form, dry form, or lyophilized form. In some embodiments, the modified siRNA and pharma- ceutically acceptable carrier and / or additive, and the drug composition, and / or siRNA complex, and any pharma- ceutically acceptable additive are essentially clean and / or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

[0461] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto in any way. EXAMPLES

[0462] Unless otherwise specified, all reagents and media used in the following examples are commercially available products, and all operations such as nucleic acid electrophoresis and real-time PCR are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0463] C57BL / 6N mice: 6-8 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., hereinafter simply referred to as C57 mice.

[0464] Unless otherwise stated, all reagent proportions provided below are calculated as volume ratios (v / v).

[0465] Unless otherwise stated, the experimental data for in vivo / in vitro effects are all

number

[0466] (Preparation Example 1) Preparation of siRNA complex L10-siXOi1M1S

[0467] In this preparation example, siRNA complex L10-siXOi1M1S was synthesized. This siRNA complex is a siRNA complex formed after L-9 complex molecule is complexed with siRNA number siXOi1M1S. The sequence of siRNA complexed in this siRNA complex is shown in Table 3.

[0468] (1-1) Synthesis of Compound L-10 Compound L-10 was synthesized according to the following method. [ka]

[0469] (1-1-1) Synthesis of Complex Terminal Segment GAL-5 [ka]

[0470] (1-1-1a) Synthesis of GAL-2 100.0g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8mmol) was dissolved in 1000ml of anhydrous pyridine, and 540ml of acetic anhydride (purchased from Enox, 5565.6mmol) was added under ice water bath, and the mixture was stirred at room temperature for 1.5 hours. The reaction liquid was poured into 10L of ice water, filtered by suction under reduced pressure, and the cake was washed with 2L of ice water. Then, acetonitrile / toluene mixed solvent (acetonitrile:toluene volume ratio = 1:1) was added until it was completely dissolved, and the solvent was evaporated to dryness to obtain 130.0g of white solid product GAL-2.

[0471] (1-1-1b) Synthesis of GAL-3 GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was dissolved in 213 ml of anhydrous 1,2-dichloroethane, and 24.0 g of TMSOTf (CAS number: 27607-77-8, purchased from Macklin Chemical, 108.0 mmol) was added under nitrogen protection conditions in an ice-water bath, and the reaction was allowed to proceed at room temperature overnight.

[0472] The reaction liquid was diluted with 400 ml of dichloromethane, filtered through diatomaceous earth, 1 L of saturated aqueous sodium bicarbonate solution was added, and the mixture was stirred uniformly. The organic phase was separated, and the aqueous phase was extracted twice with dichloroethane, each time with 300 ml of dichloroethane. The organic phases were combined and washed with 300 ml of saturated aqueous sodium bicarbonate solution and 300 ml of saturated saline solution, respectively. The organic phase was separated and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure to obtain 26.9 g of a pale yellow, viscous starch syrup-like product GAL-3.

[0473] (1-1-1c) Synthesis of GAL-4 GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane, 30 g of dried 4 Å molecular sieve powder was added, and 9.0 g of 5-hexen-1-ol (CAS number: 821-41-0, purchased from Adamas-beta, 89.9 mmol) was added and stirred at room temperature for 30 minutes. 9.08 g of TMSOTf (40.9 mmol) was added under ice bath and nitrogen protection, and the reaction was allowed to proceed with stirring at room temperature overnight. The 4Å molecular sieve powder was removed by filtration, the filtrate was diluted with 300ml of dichloromethane, filtered through diatomaceous earth, 500ml of saturated aqueous sodium bicarbonate was added, and the mixture was stirred and washed for 10 minutes. The organic phase was separated, and the aqueous phase was extracted once with 300ml of dichloroethane. The organic phases were combined and washed with 300ml of saturated aqueous sodium bicarbonate and 300ml of saturated saline, respectively. The organic phase was separated and dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 41.3g of yellow starch syrup-like product GAL-4, which was directly used for the next oxidation reaction without purification.

[0474] (1-1-1d) Synthesis of GAL-5 GAL-4 (14.9 g, 34.7 mmol) obtained by the method described in step (1-1-1c) was dissolved in a mixed solvent of 77 ml of dichloromethane and 77 ml of acetonitrile, and 103 ml of deionized water and 29.7 g of sodium periodate (CAS number: 7790-28-5, purchased from Aladdin, 138.8 mmol) were added, and the mixture was stirred for 10 minutes in an ice-water bath, and ruthenium (III) chloride (CAS number: 14898-67-0, purchased from Energy, 238 mg, 1.145 mmol) was added, and the mixture was reacted at room temperature overnight. The reaction solution was diluted with 300 ml of water and stirred, and the pH was adjusted to about 7.5 by adding saturated sodium bicarbonate, and the organic phase was separated and discarded, and the aqueous phase was extracted with dichloromethane three times, 200 ml each time, and the organic phase was discarded. The pH of the aqueous phase was adjusted to about 3 with solid citric acid, and extracted three times with dichloromethane, each time with 200 ml. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 6.85 g of a white foamy solid product, GAL-5. 1 H NMR (400 MHz,DMSO) δ 12.01 (br,1H),7.83 (d,J = 9.2 Hz,1H),5.21 (d,J = 3.2 Hz,1H),4.96 (dd,J = 11.2,3.2 Hz,1H),4.49 (d,J = 8.4 Hz,1H),4.07-3.95 (m,3H),3.92-3.85 (m,1H),3.74-3.67 (m,1H),3.48-3.39 (m,1H),2.20 (t,J = 6.8 Hz,2H),2.11 (s,3H),2.00 (s,3H),1.90 (s,3H),1.77 (s,3H),1.55-1.45 (m,4H).

[0475] (1-1-2) Synthesis of L-8 [ka]

[0476] J-0 (9.886 g, 52.5 mmol, purchased from Alfa Acer Inc.) and GAL-5 (72.819 g, 162.75 mmol, a combination of products from multiple lots) obtained in step (1-1-1) were dissolved in 525 ml of dichloromethane, and diisopropylethylamine (DIEA, 44.782 g, 346.50 mmol), benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate / The ester (PyBOP, 90.158 g, 173.25 mmol) and hydroxybenzotriazole (HOBt, 23.410 g, 173.25 mmol) were added, and the mixture was reacted at room temperature for 4 h. 20 ml of saturated sodium bicarbonate and 200 ml of saturated saline were added to wash the mixture. The aqueous phase was extracted twice with dichloromethane, 100 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure to obtain the crude product. For purification, 200-300 mesh normal phase silica gel was used, the acidity of the silica gel was neutralized with 10 wt% triethylamine, the column was equilibrated with 1 wt‰ triethylamine, and gradient elution was performed with dichloromethane:methanol = 100:25-100:40. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 38.8 g of pure L-8. 1 H NMR (400 MHz,DMSO) δ 7.84 (d,J = 9.0 Hz,3H),7.27-7.23 (m,1H),7.13-7.18 (m,1H),5.22 (d,J = 3.1 Hz,3H),4.97 (dd,J = 11.3,3.1 Hz,3H),4.48 (d,J = 8.4 Hz,3H),4.09-3.98 (m,9H),3.88 (dd,J = 19.3,9.3 Hz,3H),3.75-3.66 (m,3H),3.44-3.38 (m,3H),3.17-3.30 (m,4H),3.10-2.97 (m,4H),2.35-2.20 (m,6H),2.15-2.08 (m,9H),2.07-1.98 (m,13H),1.94-1.87 (m,9H),1.81-1.74 (m,9H),1.65-1.42 (m,18H). MS m / z:C 85 H 119 N 7 O 30 , [M+H]+ , theoretical value: 1477.59, measured value: 1477.23.

[0477] (1-1-3a) Synthesis of A-1 [ka]

[0478] Dissolve DMTrCl (4,4'-bismethoxytrityl chloride, 101.65g, 300mmol) in 1000ml of anhydrous pyridine, add DL-calcium glycerate hydrate (28.63g, 100mmol), react at 45℃ for 20h, filter the reaction solution, rinse the cake with 200ml of DCM, evaporate the filtrate under reduced pressure to dryness, dissolve the residue in 500ml of dichloromethane again, wash with 0.5M triethylamine phosphate (pH=7~8) twice with 200ml each time, extract the aqueous phase with dichloromethane twice with 200ml each time, combine the organic phases, and wash with anhydrous sodium sulfate. The mixture was dried, filtered, and the solvent was evaporated to dryness under reduced pressure. The mixture was purified on a 200-300 mesh normal phase silica gel column, and gradient eluted with petroleum ether:ethyl acetate:dichloromethane:methanol=1:1:1:0.35-1:1:1:0.55. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure. The product was dissolved again in 600ml of dichloromethane, and washed once with 200ml of 0.5M triethylamine phosphate. The aqueous phase was extracted once with 200ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure. The mixture was vacuumed overnight with a vacuum oil pump to obtain 50.7g of white solid product A-1. 11H NMR (400 MHz, DMSO-d6) δ 7.46 (ddd, J = 6.5, 2.3, 1.1 Hz, 1H), 7.40 - 7.28 (m, 7H), 6.89 - 6.81 (m, 4H), 4.84 (d, J = 5.0 Hz, 1H), 4.36 - 4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J = 12.4, 7.0 Hz, 1H), 3.67 (dd, J = 12.3, 7.0 Hz, 1H), 2.52 (q, J = 6.3 Hz, 6H), 1.03 (t, J = 6.3 Hz, 9H). MS m / z: C 24 H 23 O 6 , [M - H] - , Theoretical value: 407.15, Measured value: 406.92.

[0479] (1 - 1 - 3b) Synthesis of L - 7

Chemical Structure

[0480] L-8 (40 g, 27.09 mmol, a combination of products from multiple lots) obtained in step (1-1-2) and A-1 (41.418 g, 81.27 mmol) obtained in step (1-1-3a) were mixed and dissolved in 271 ml of dichloromethane, and 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (24.318 g, 81.37 mmol) was added, and diisopropylethylamine (2 The organic phase was washed with 800ml of saturated sodium bicarbonate, the aqueous phase was extracted with dichloromethane three times, each time with 50ml, the organic phase was washed with 150ml of saturated saline, the aqueous phase was extracted once with 50ml of dichloromethane, the organic phase was combined, dried with anhydrous sodium sulfate, filtered, the solvent was evaporated to dryness under reduced pressure, and the crude product was obtained by foaming and drying overnight with a vacuum oil pump. For column purification, 2 kg of 200-300 mesh normal phase silica gel was used, the acidity of the silica gel was neutralized with 200 ml of triethylamine, the column was equilibrated with petroleum ether containing 1 wt% triethylamine, and gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6, the product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 40.4 g of pure L-7. 1 H NMR (400 MHz,DMSO) δ7.90-7.78 (m,4H),7.75-7.64 (m,1H),7.38-7.18 (m,9H),6.91-6.83 (m,4H),5.25-5.10 (m,4H),4.97 (dd,J = 11.2,3.2 Hz,3H),4.48-4.30 (m,4H),4.02 (s,9H),3.93-3.84 (m,3H),3.76-3.66 (m,9H),3.45-3.35 (m,3H),3.24-2.98 (m,10H),2.30-2.20 (m,2H),2.11-1.88 (m,31H),1.80-1.40 (m, 28H). MS m / z: C 90 H 128 N 7 O 35 , [M-DMTr] + , theoretical value: 1564.65, actual value: 1564.88.

[0481] Synthesis of (1-1-4)L-9 [ka]

[0482] L-7 (40 g, 21.4247 mmol) obtained in step (1-1-3b), succinic anhydride (4.288 g, 42.8494 mmol) and 4-dimethylaminopyridine (DMAP, 5.235 g, 42.8494 mmol) were mixed and dissolved in 215 ml of dichloromethane, diisopropylethylamine (DIEA, 13.845 g, 107.1235 mmol) was further added, and the mixture was stirred at 25°C for 24 hours. The reaction solution was washed with 800 ml of 0.5 M triethylamine phosphate, and the aqueous phase was extracted three times with 5 ml of dichloromethane each time. The organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. For column purification, 1 kg of 200-300 mesh normal phase silica gel was used, the acidity of the silica gel was neutralized with 1 wt% triethylamine, the column was equilibrated with dichloromethane, and gradient elution was performed with dichloromethane:methanol = 100:18-100:20 containing 1 wt% triethylamine. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 31.0 g of pure L-9 complex molecule. 1H NMR (400 MHz,DMSO) δ 8.58 (d,J = 4.2 Hz,1H),7.94-7.82 (m,3H),7.41-7.29 (m,5H),7.22 (d,J = 8.1 Hz,5H),6.89 (d,J = 8.3 Hz,4H),5.49-5.37 (m,1H),5.21 (d,J = 3.0 Hz,3H),4.97 (d,J = 11.1 Hz,3H),4.49 (d,J = 8.2 Hz,3H),4.02 (s,9H),3.88 (dd,J = 19.4,9.4 Hz,3H),3.77-3.65 (m,9H),3.50-3.39 (m,6H),3.11-2.90 (m,5H),2.61-2.54 (m,4H),2.47-2.41 (m,2H),2.26-2.17 (m,2H),2.15-1.95 (m,22H),1.92-1.84 (m,9H),1.80-1.70 (m,10H),1.65-1.35 (m,17H),1.31-1.19 (m,4H),0.96 (t,J = 7.1 Hz,9H). MS m / z:C 94 H 132 N 7 O 38 , [M-DMTr] + , theoretical value: 1664.72, actual value: 1665.03.

[0483] (1-1-5) Synthesis of Compound L-10 [ka]

[0484] In this process, the L-10 compound was prepared by binding the L-9 conjugate molecule to a solid support.

[0485] The L-9 complex molecule (22.751 g, 11 mmol) obtained in step (1-1-4), O-benzotriazole-tetramethyluronium hexafluorophosphate / ester (HBTU, 6.257 g, 16.5 mmol) and diisopropylethylamine (DIEA, 2.843 g, 22 mmol) were mixed and dissolved in 900 ml of acetonitrile. The mixture was stirred at room temperature for 5 minutes, and the reaction solution was added with aminomethyl resin (88 g, 100-200 mesh, amino group loading amount 40 0μmol / g, purchased from Nankaiwasei Co., Ltd.) was added, and the reaction was carried out on a shaker at 25 ° C., and the reaction was carried out at a rotation speed of 150 rpm for 18 h, followed by filtration, and the cake was rinsed twice with 300 ml each time with DCM, rinsed three times with 300 ml each time with acetonitrile, and dried with a vacuum oil pump for 18 h, and then the raw materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) were added according to the composition ratio shown in Table 2 to carry out the capping reaction. The mixture was placed on a shaker at 25 ° C., and the reaction was carried out at a rotation speed of 150 rpm for 5 h, and the reaction solution was filtered, and the cake was rinsed three times with 300 ml each time with acetonitrile, and the solvent was evaporated under reduced pressure until dry, and dried under reduced pressure overnight with a vacuum oil pump to obtain 102 g of L-10 compound (i.e., L-9 complex molecule bound to a solid phase support) with a loading of 90.8 μmol / g.

[0486] [Table 2] Here, CapA and CapB are capping reagent solutions, CapA is a 20% by volume solution of N-methylimidazole in a pyridine / acetonitrile mixture with a volume ratio of pyridine to acetonitrile of 3:5, and CapB is a 20% by volume solution of acetic anhydride in acetonitrile.

[0487] (1-2) Synthesis of the sense strand of siRNA complex L10-siXOi1M1S Using the solid-phase phosphoramidite method, the L-10 compound prepared in the above step was circulated as a starting material, and nucleoside monomers were bound one by one in the 3'-5' direction according to the order of nucleotides in the sense strand. Each time a nucleoside monomer was bound, four reactions were carried out: deprotection, coupling, capping, oxidation, or sulfurization. When two nucleotides were bound by a phosphate ester, four reactions were carried out when the next nucleoside monomer was bound: deprotection, coupling, capping, and oxidation. When two nucleotides were bound by a thiophosphate ester, four reactions were carried out when the next nucleoside monomer was bound: protection, coupling, capping, and sulfurization. The synthesis conditions were specified as follows.

[0488] The nucleoside monomers were provided in acetonitrile at a concentration of 0.1 M, and the conditions for each deprotection reaction were the same, i.e., the temperature was 25° C. and the reaction time was 70 s. The deprotection reagent was a solution of dichloroacetic acid in dichloromethane (3% v / v), and the molar ratio of dichloroacetic acid to the 4,4′-dimethoxytrityl protecting group on the solid support was 5:1.

[0489] The coupling reaction conditions were the same for all of them: temperature 25°C, molar ratio of nucleic acid sequence to be bound to solid support to nucleoside monomer 1:10, molar ratio of nucleic acid sequence to be bound to solid support to coupling reagent 1:65, reaction time 600 seconds, and coupling reagent 0.5M acetonitrile solution of 5-(Ethylthio)-1H-tetrazole (ETT).

[0490] The capping conditions were the same for all of the samples, with a temperature of 25° C. and a reaction time of 15 seconds. The capping reagent solution was a mixed solution of CapA and CapB in a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence to be bound to the solid phase carrier was acetic anhydride:N-methylimidazole:nucleic acid sequence to be bound to the solid phase carrier=1:1:1.

[0491] The oxidation reaction conditions were the same for each reaction: temperature 25°C, reaction time 15 seconds, and oxidation reagent 0.05M iodine water. The molar ratio of iodine to the nucleic acid sequence bound to the solid support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0492] The conditions for each sulfurization reaction were the same: temperature 25°C, reaction time 300 seconds, and sulfurization reagent xanthan hydride. The molar ratio of sulfurization reagent to the nucleic acid sequence to be bound to the solid support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile:pyridine = 1:1.

[0493] After the coupling of the last nucleoside monomer was completed, the nucleic acid sequence bound to the solid support in turn was cleaved, deprotected, purified, desalted, and then lyophilized to obtain the sense strand.

[0494] The cleavage and deprotection conditions were as follows: the synthesized carrier-bound nucleotide sequence was added to ammonia water with a concentration of 25 wt%, the dosage of the ammonia water was 0.5 ml / μmol, and reacted at 55°C for 16 h, the remaining carrier was filtered off, and the supernatant was vacuum concentrated to dryness.

[0495] The purification and desalting conditions are as follows. The purification of nucleic acids was completed by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent A: 20 mM sodium phosphate (pH 8.1), solvent water / acetonitrile = 9: 1 (volume ratio), eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent water / acetonitrile = 9: 1 (volume ratio), elution gradient: eluent A: eluent B = 100: 0 to 50: 50 gradient elution. The product eluate was collected and then combined, and desalted using a reverse phase chromatography purification column, and the specific conditions were desalted using a dextran gel column, the packing material was dextran gel G25 (Sephadex G25), and eluted with deionized water.

[0496] The detection method is as follows. The purity of the sense strand was detected using ion exchange chromatography (IEX-HPLC), and the molecular weight was analyzed by liquid chromatography mass spectrometry (LC-MS). The theoretical value was 7584.5, and the actual value was 7584.0. The actual value was consistent with the theoretical value, indicating that a sense strand SS conjugated to the L-9 conjugate molecule at the 3' end was synthesized.

[0497] (1-3) Synthesis of antisense strand of siRNA complex L10-siXOi1M1S The solid-phase phosphoramidite method was used to prepare a standard solid-phase support (UnyLinker TM The antisense strand of the siRNA complex L10-siXOf1M1S was synthesized by circulating loaded NittoPhase® HL Solid Supports (Kinovate Life Sciences). The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid phase synthesis method were the same as those in the synthesis of the sense strand. The antisense strand AS was obtained by lyophilization.

[0498] The purity of the antisense strand was detected by ion exchange chromatography (IEX-HPLC), and the molecular weight of the antisense strand was analyzed by liquid chromatography-mass spectrometry (LC-MS). The measured values ​​were consistent with the theoretical values, indicating that the antisense strand AS having the desired sequence was synthesized.

[0499] (1-4) Synthesis of siRNA complex L10-siXOi1M1S For the siRNA complex L10-siXOi1M1S, the sense strand and the antisense strand were dissolved in water for injection, respectively, to obtain a 40 mg / mL solution, mixed in an equimolar ratio, heated at 50°C for 15 min, cooled at room temperature, and then annealed to obtain a product, which was freeze-dried to obtain a freeze-dried powder. The siRNA complex was diluted with ultrapure water (Milli-Q ultrapure water device, resistivity 18.2 MΩ*cm (25°C)) to a concentration of 0.2 mg / mL, and then the molecular weight was detected by a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). The actual measured value was consistent with the theoretical value, indicating that the synthesized siRNA complex was a double-stranded nucleic acid sequence of the intended design containing the L-9 complex molecule. Its structure is shown in formula (403). The siRNA was a sequence corresponding to the siRNA complex L10-siXOi1M1S shown in Table 3.

[0500] [Table 3] In the above, capital letters C, G, U, and A represent the base sequence of nucleotides, lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and lower case letter s represents that the two nucleotides on the left and right of the letter s are bonded by a thiophosphate group.

[0501] (Preparation Example 2 and Comparative Preparation Example 3) Further, siRNA complex L10-siXOk1M1S and comparative siRNA complex NC were synthesized according to the method of Preparation Example 1, except that the sense and antisense strands corresponding to L10-siXOk1M1S and NC in Table 3 were used instead of the sense and antisense strand sequences of siRNA complex L10-siXOi1M1S. The siRNAs contained in these siRNA complexes have sense and antisense strands corresponding to L10-siXOk1M1S and NC in Table 3, respectively.

[0502] After the preparation was completed, the molecular weights of the prepared siRNA complexes L10-siXOk1M1S and NC were detected according to the method of Preparation Example 1, and the measured values ​​were consistent with the theoretical values, indicating that the synthesized siRNA complexes were double-stranded nucleic acid sequences of the intended design containing L-9 complex molecules. Their structures are shown in formula (403). The siRNAs contained in these siRNA complexes were sequences corresponding to the siRNA complexes L10-siXOk1M1S and NC shown in Table 3, respectively.

[0503] (Preparation Examples 4 to 18 and Comparative Preparation Example 19) <Synthesis of siRNA provided by the present disclosure> The sense strand or antisense strand of the siRNA sequence shown in Table 4 was synthesized using solid phase synthesis, and equimolar amounts of the complementary sense strand and antisense strand in Table 4 were dissolved using DEPC-treated water, followed by annealing to obtain siXOa1M1S, siXOb1M1S, siXOc1M1S, siXOd1M1S, siXOe1M1S, siXOf1M1S, siXOg1M1S, siXOh1M1S, siXOi1M1S, siXOj1M1S, siXOk1M1S, siXOl1M1S, siXOa0, siXOe0 and siXOf0 provided by the present disclosure, as well as comparative siRNA CON-siXOf, as shown in Table 4.

[0504] [Table 4] In the above, capital letters C, G, U, and A represent the base sequence of nucleotides, lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide, lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide, and lower case letter s represents that the two nucleotides on the left and right of the letter s are bonded by a thiophosphate group.

[0505] In the above sequence preparation process, if the target sequence contains unmodified nucleotides, the product is treated with ammonia water under cleavage and deprotection conditions, and then dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride are added to remove the 2'-TBDMS protection of ribose.

[0506] After the preparation of the siRNA or siRNA complex was completed, it was freeze-dried and stored as a solid powder. When used, it could be dissolved again in a solution of the desired concentration using, for example, water for injection, physiological saline (NS), phosphate buffer (PB), or phosphate buffer solution (PBS), etc.

[0507] Experimental Example 1 In vitro suppression activity of the disclosed siRNA HEK293A cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in H-DMEM complete medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 0.2% by volume of penicillin-streptomycin (Gibco, Invitrogen) at 37 °C for 24 h at 4 °C under 5% CO. 2 The cells were cultured at 37°C in an incubator containing 95% air.

[0508] According to the method described in Kumico Ui-Tei et.al., Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Research, 2008.36(7), 2136-2151, a test plasmid was constructed, and the siRNAs to be evaluated (siXOa0, siXOe0, siXOf0 and CON-siXOf) were transfected into HEK293A cells, and the suppression activity of the siRNA was reflected from the expression level of the dual luciferase reporter gene. The specific steps are as follows.

[0509] [1] Construction of test plasmids psiCHECK TM -2(Promega TM ) plasmid was used to construct test plasmids containing siRNA target sequences of interest. The target sequences for the siRNAs to be evaluated are shown below. Target sequence of siXOa0: GAGATGAAGTTCAAGAATA (SEQ ID NO: 731) Target sequence of siXOe0: ACATGGACAACTGCTATAA (SEQ ID NO: 732) Destination sequence for siXOf0: TAGCAAGCTCTCAGTATCA (SEQ ID NO: 733) Target sequence of CON-siXOf: CTAGCAAGCTCTCAGTATC (SEQ ID NO: 734)

[0510] psiCHECK the target sequence TM The fragment was cloned into the Xho I / Not I sites of the -2 plasmid.

[0511] [2] Transfection 8 × 10 HEK293A cells 3 Cells were seeded into a 96-well plate at 100 cells / well. After 16 h, when the cell growth density reached 70-80%, the complete H-DMEM medium in the culture wells was aspirated off, and 80 μl of Opti-MEM medium (GIBCO) was added per well. Culture was continued for 1.5 h.

[0512] For each siRNA, the corresponding test plasmid was diluted with DEPC-treated water to prepare a test plasmid standard solution of 200 ng / μl. For each siRNA, siRNA standard solutions with concentrations (as siRNA) of 10 nM, 3 nM, and 1 nM were prepared using siRNA and DEPC-treated water, respectively.

[0513] For each siRNA, 1A1 solution was prepared containing 1 μl of 10 nM siRNA dilution standard solution, 0.05 μl of test plasmid dilution standard solution (containing 10 ng of test plasmid), and 10 μl of Opti-MEM medium.

[0514] For each siRNA, 1A2 solution was prepared containing 1 μl of 3 nM siRNA dilution standard solution, 0.05 μl of test plasmid dilution standard solution (containing 10 ng of test plasmid), and 10 μl of Opti-MEM medium.

[0515] For each siRNA, 1A3 solution was prepared containing 1 μl of siRNA dilution standard solution with a concentration of 1 nM, 0.05 μl of test plasmid dilution standard solution (containing 10 ng of test plasmid), and 10 μl of Opti-MEM medium.

[0516] 0.2 μl Lipofectamine TM A 1B solution was made containing 2000 and 10 μl of Opti-MEM medium.

[0517] A 1C solution containing 0.05 μl of the test plasmid dilution standard solution (containing 10 ng of the test plasmid) and 10 μl of Opti-MEM medium was prepared.

[0518] For each siRNA, one 1B solution was mixed with one 1A1 solution, one 1A2 solution, and one 1A3 solution, and incubated at room temperature for 20 min to obtain transfection complexes 1X1, 1X2, and 1X3, respectively. One 1B solution was mixed with one 1C solution, and incubated at room temperature for 20 min to obtain transfection complex 1X4.

[0519] For each siRNA, the transfection complex 1X1 was added to three culture wells at a volume of 20 μl / well, respectively, and mixed evenly to obtain a co-transfection mixture containing siRNA with a final siRNA concentration of about 0.1 nM, which was designated as test group 1.

[0520] For each siRNA, transfection complex 1X2 was added to another three culture wells at a volume of 20 μl / well, respectively, and mixed evenly to obtain a co-transfection mixture containing siRNA with a final siRNA concentration of about 0.03 nM, which was designated as test group 2.

[0521] For each siRNA, the transfection complex 1X3 was added to another three culture wells at a volume of 20 μl / well, respectively, and mixed evenly to obtain a co-transfection mixture containing siRNA at a final concentration of 0.01 nM, which was designated as test group 3.

[0522] To another three culture wells, transfection complex 1X4 was added at a dosage of 20 μl / well, respectively, to obtain a transfection mixture without siRNA, which served as a control group.

[0523] The siRNA-containing and siRNA-free cotransfection mixtures were cotransfected in the culture wells for 4 h, after which 100 μl of H-DMEM complete medium containing 20% ​​FBS was added per well. The 96-well plate was incubated at 4 °C for 2 h at 4 °C for 1 h. 2 The cells were then placed in an incubator and cultured for 24 hours.

[0524] [3] Detection The medium in the culture wells was removed by suction, and 150 μl of a mixture of Dual-Glo® Luciferase Reagent and H-DMEM complete medium (volume ratio 1:1) was added to each well, mixed thoroughly until homogenous, and incubated at room temperature for 10 minutes. Then, 120 μl of the mixture was transferred to a 96-well ELISA plate, and the Firefly chemiluminescence value (Fir) was read using a Synergy II multifunction microplate reader (BioTek). Then, 60 μl of Dual-Glo® Stop & Glo (registered trademark) reagent was added, thoroughly mixed until homogenous, and incubated at room temperature for 10 minutes. The chemiluminescence value (Ren) of Renilla was then read using a microplate reader according to the arrangement of the read Fir.

[0525] The emission ratio Ratio = Ren / Fir of each well was calculated, and the emission ratio Ratio (test) or Ratio (control) of each test group or control group was the average of the Ratios of three culture wells. The emission ratio of each test group was normalized based on the emission ratio of the control group to obtain the ratio Ratio (test) / Ratio (control), which represents the expression level of the Renilla reporter gene, i.e., the residual activity. The inhibition rate of the target sequence = (1-R) ​​x 100%.

[0526] Table 5 shows the results of the inhibitory activity against the target sequence by the siRNAs to be evaluated at different concentrations.

[0527] [Table 5]

[0528] As can be seen from the results, the siRNA of the present disclosure exhibits excellent in vitro suppression activity against the target sequence at each concentration, and also exhibits concentration dependency.In particular, at an siRNA concentration of 0.1nM, the target sequence suppression rate is at least 61.39%, and in particular, siXOf exhibits a target sequence suppression rate of 68.79% at a concentration of 0.03nM, and the target sequence suppression rate at a concentration of 0.1nM reaches a high of 85.43%.In stark contrast, although the sequence of comparative siRNA CON-siXOf is very similar to that of siXOf, even at a concentration of 0.1nM, the target sequence suppression rate is only 48.24%, revealing that the siRNA of the present disclosure unexpectedly exhibits a good XO gene expression suppression effect.

[0529] Experimental Example 2 IC of siRNA against XO mRNA in CAL-27 cells 50 Detection of CAL-27 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in H-DMEM complete medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 0.2% by volume of penicillin-streptomycin (Gibco, Invitrogen) at 37 °C for 24 h at 4 °C under 5% CO. 2 The cells were cultured at 37°C in an incubator containing 95% air.

[0530] 7.5 × 10 CAL-27 cells 4 Cells were seeded on a 24-well plate at 100 cells / well. After 16 h, when the cell growth density reached 70-80%, the H-DMEM complete medium in the culture wells was aspirated off, and 500 μl of Opti-MEM medium (GIBCO) was added to each well and cultured for 1.5 h. The cells were washed with HBSS solution, mixed uniformly, and then resuspended at 6 × 10 5 Cells / well were seeded into 96-well plates, with the inoculum volume being 45 μl / well.

[0531] Using DEPC-treated water, a total of eight siRNA dilution standard solutions were prepared from each of the siRNAs siXOa1M1S, siXOb1M1S, siXOc1M1S, siXOd1M1S, siXOe1M1S, and siXOf1M1S, with concentrations of 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM, 1.44 nM, and 0.72 nM (as siRNA).

[0532] The above 8 different concentrations of siRNA dilution standard solution were added in sequence at 15μL / well to different culture wells inoculated with the above CAL-27 cells. Thus, for each of the above siRNAs, the final siRNA concentrations in each culture well were 5μM, 1μM, 0.2μM, 0.04μM, 0.008μM, 0.0016μM, 0.32nM, and 0.064nM, respectively, and mixed uniformly to form the test group. The culture wells inoculated with only CAL-27 cells and not added with the siRNA dilution standard solution were used as the control group.

[0533] The test and control groups were electroporated using an electroporator (EBXP-H1, Ichida Cell Electroporator), and the transfection parameters were as follows: voltage: 210 V, pulse duration: 100 μs, number of pulses: 6, pulse interval: 1000 ms.

[0534] 240μl of H-DMEM complete medium containing 20% ​​FBS was added to each culture well of the above-mentioned transfected test group and control group samples, to obtain a transfected cell culture solution. For each culture well, 140μl of the transfected cell culture solution was transferred to two culture wells of a 24-well plate, and 855μl of H-DMEM complete medium containing 20% ​​FBS was added to each culture well of the 24-well plate to which the cell culture solution was added, and the culture was continued for 24h to obtain a test cell culture solution. Then, using RNAVzol (purchased from Vigorous Biotechnology (Beijing) Co., Ltd., No. N002), the total RNA in the test cell culture solution of each well was extracted according to the steps described in the instruction manual.

[0535] 1 μg of total RNA was taken from each well of a 24-well plate and reverse-transcribed using the Goldenstar Reverse Transcription Kit. TM The reagents provided by the RT6 cDNA Synthesis Kit (purchased from Beijing Keike Xinye Biotechnology Co., Ltd., No. TSK301M) were used. TM Oligo (dT) 17 was selected as a primer, 20 μl of the reverse transcription reaction system was arranged according to the reverse transcription operation steps in the kit's manual, and reverse transcription was performed on the total RNA of the cells. As the conditions for reverse transcription, the reverse transcription reaction system was incubated at 50°C for 50 min, then at 85°C for 5 min, and finally at 4°C for 30 s. After the reaction was completed, 80 μl of DEPC-treated water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0536] For each reverse transcription reaction system, 5 μl of the solution containing the above cDNA was used as a template, and 20 μl of qPCR reaction system was arranged using the reagents provided by NovoStart® SYBR qPCR SuperMix Plus kit (purchased from Kinki Protein Technology Co., Ltd., No. E096-01B). Here, the PCR primer sequences for amplifying the target gene XO and the endogenous reference gene GAPDH are as shown in Table 7. The final concentration of each primer was 0.25 μM. Each qPCR reaction system was placed in an ABI StepOnePlus Real-Time PCR device and amplification was performed in a three-step method, and the amplification procedure was performed by pre-denaturing at 95°C for 10 minutes, denaturing at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and elongating at 72°C for 30 seconds. After repeating the above denaturation, annealing, and elongation processes a total of 40 times, a product W containing the amplified target gene XO and the endogenous reference gene GAPDH was obtained. Then, product W was incubated at 95°C for 1 min, at 55°C for 30 s, and at 95°C for 30 s, and melting curves of the target gene XO and the endogenous reference gene GAPDH in product W were obtained using a real-time fluorescent quantitative PCR device, and the Ct values ​​of the target gene XO and the endogenous reference gene GAPDH were obtained.

[0537] [Table 6]

[0538] Using the comparative Ct (ΔΔCt) method, relative quantitative calculation was performed for the expression level of the target gene XO in each test group and the control group. The calculation method is as follows: ΔCt(test group)=Ct(target gene in test group)-Ct(endogenous reference gene in test group) ΔCt(control group)=Ct(gene of interest in control group)-Ct(endogenous reference gene in control group) ΔΔCt(test group)=ΔCt(test group)-ΔCt(average of control group) ΔΔCt(control group) = ΔCt(control group) - ΔCt(average of control group) Here, ΔCt(control group mean) is the arithmetic mean of ΔCt(control group) for each of the two culture wells in the control group. Therefore, each culture well in the test group and the control group corresponds to one ΔΔCt value.

[0539] The expression levels of XO mRNA in the test groups were normalized to that in the control group, and the expression level of XO mRNA in the control group was defined as 100%.

[0540] Relative expression level of XO mRNA in the test group = 2 -ΔΔCt(試験群) ×100%.

[0541] For the same test group of siRNA, the average relative expression level of XO mRNA in the test group at each concentration is the arithmetic mean of the relative expression levels of the two culture wells at that concentration.

[0542] The dose-effect curves were fitted using the log(inhibitor) vs. response-variable slope function of Graphpad 6.0 software, and the IC 50 Specifically, the fitted dose-effect curve satisfies the following formula:

[0543]

number

[0544] 1A-1F are dose-effect curves fitted according to the relative expression levels of XO mRNA in CAL-27 cells in vitro after transfection with siXOa1M1S, siXOb1M1S, siXOc1M1S, siXOd1M1S, siXOe1M1S and siXOf1M1S, respectively. From the dose-effect curves and the corresponding formula, the corresponding X for Y=50% was calculated. 50 Determine IC values ​​for each siRNA 50 Value=10^X 50 (nM) was calculated.

[0545] IC of each siRNA against XO mRNA 50 The values ​​are summarized in Table 7.

[0546] [Table 7]

[0547] As can be seen from Table 7, the siRNAs provided by the present disclosure exhibited high XO mRNA suppression activity in CAL-27 cells in vitro and IC 50 was 0.037-0.3277 μM.

[0548] Experimental Example 3 Measurement of the suppression rate of XO mRNA by siRNA in mouse primary hepatocytes Primary mouse hepatocytes were extracted from fresh liver tissue of normal C57BL / 6N mice, inoculated onto tissue culture dishes coated with type I collagen, and cultured in RPMI 1460 medium containing 1x penicillin-streptomycin and 10% FBS at 5% CO. 2 The plates were cultured for 30 minutes at 37°C in a 95% air-containing incubator.

[0549] Discard the medium and culture the primary mouse hepatocytes in opti-MEM at a density of 1 × 10 6The volume of the mouse primary hepatocyte suspension was adjusted to 0.5 mL / well, and the number of mouse primary hepatocytes was 5 × 10 4 cells / well.

[0550] Using DEPC-treated water, 20 μM siRNA dilution standard solutions were prepared for each of siXOg1M1S, siXOh1M1S, siXOi1M1S, siXOj1M1S, siXOk1M1S and siXOl1M1S siRNA.

[0551] A 1A solution was prepared. A 1A solution was prepared for each siRNA. Each 1A solution contained 0.6 μl of the above siRNA dilution standard solution and 50 μl of Opti-MEM medium, in that order.

[0552] Prepare 1B solution. Add 1 μl of Lipofectamine to each 1B solution. TM 2000 and 50 μl of Opti-MEM medium.

[0553] One of the 1B solutions was mixed with the obtained 1A solution of each siRNA, and each was incubated at room temperature for 20 minutes to obtain 1X transfection complexes of each siRNA.

[0554] One 1B solution and 50 μl of Opti-MEM medium were mixed and incubated at room temperature for 20 minutes to obtain transfection complex 1X'.

[0555] 1X transfection complex of each siRNA was added to each culture well at a volume of 100 μl / well and mixed evenly to obtain a transfection complex with a final concentration of each siRNA of approximately 20 nM. Three culture wells were transfected with 1X transfection complex of each siRNA to obtain a transfection mixture containing siRNA, which was used as the test group.

[0556] To another three culture wells, transfection complex 1X' was added at a dosage of 100 μl / well, respectively, to obtain a transfection mixture without siRNA, which served as a blank control group.

[0557] The transfection mixtures containing each siRNA and the transfection mixtures not containing siRNA were transfected into different culture wells for 4 h, and then 1 ml of H-DMEM complete medium containing 20% ​​FBS was added to each well. The 24-well plate was incubated at 4 °C for 3 h at 4 °C for 1 h. 2 The cells were then placed in an incubator at 37°C and cultured for 24 hours.

[0558] Then, total RNA was extracted from the cells in each well using RNAVzol (purchased from Vigorous Biotechnology (Beijing) Co., Ltd., No. N002) according to the method described in the instruction manual.

[0559] 1 μg of total RNA was taken from each well of cells and reverse-transcribed using the Goldenstar TM The reagents provided by the RT6 cDNA Synthesis Kit (purchased from Beijing Keike Xinye Biotechnology Co., Ltd., No. TSK301M) were used. TM Oligo (dT) 17 was selected as a primer, 20 μl of the reverse transcription reaction system was placed according to the reverse transcription operation steps in the kit's instruction manual, and reverse transcription was performed on the total RNA in the cells of each well. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the reverse transcription reaction system was incubated at 50°C for 50 min, then at 85°C for 5 min, and finally at 4°C for 30 s. After the reaction was completed, 80 μl of DEPC-treated water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0560] For each reverse transcription reaction system, 5 μl of the solution containing the above cDNA was used as a template, and 20 μl of qPCR reaction system was arranged using the reagents provided by NovoStart® SYBR qPCR SuperMix Plus kit (purchased from Kinki Protein Technology Co., Ltd., No. E096-01B). Here, the PCR primer sequences for amplifying the target gene XO and the endogenous reference gene GAPDH are shown in Table 7. The final concentration of each primer was 0.25 μM. Each qPCR reaction system was placed in an ABI StepOnePlus Real-Time PCR device and amplification was performed in a three-step method, and the amplification procedure was performed by pre-denaturing at 95°C for 10 minutes, denaturing at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and elongating at 72°C for 30 seconds. After repeating the above denaturation, annealing, and elongation processes a total of 40 times, a product W containing the amplified target gene XO and the endogenous reference gene GAPDH was obtained. Then, product W was incubated at 95°C for 15 s, at 60°C for 1 min, and at 95°C for 15 s, and the melting curves of the target gene XO and the endogenous reference gene GAPDH in product W were obtained using a real-time fluorescent quantitative PCR device, and the Ct values ​​of the target gene XO and the endogenous reference gene GAPDH were obtained.

[0561] [Table 8]

[0562] The comparative Ct (ΔΔCt) method was used to perform relative quantitative calculations for the target gene XO in each test group, and the calculation method is as follows: ΔCt(test group)=Ct(target gene in test group)-Ct(endogenous reference gene in test group) ΔCt(control group)=Ct(gene of interest in control group)-Ct(endogenous reference gene in control group) ΔΔCt(test group)=ΔCt(test group)-ΔCt(average of control group) ΔΔCt(control group) = ΔCt(control group) - ΔCt(average of control group) Here, ΔCt(control mean) is the arithmetic mean of ΔCt(control) for each of the three culture wells in the control group. Therefore, each culture well in both the test and control groups corresponded to one ΔΔCt value.

[0563] The expression levels of XO mRNA in the test groups were normalized based on the control group, and the expression level of XO mRNA in the blank control group was defined as 100%.

[0564] Relative expression level of XO mRNA in the test group = 2 -ΔΔCt(試験群) ×100%.

[0565] The XO mRNA suppression rate of the test group = (1 - relative expression level of XO mRNA of the test group) x 100%.

[0566] FIG. 2 is a histogram of the relative expression level of XO mRNA in mouse primary hepatocytes after transfection with siXOg1M1S, siXOh1M1S, siXOi1M1S, siXOj1M1S, siXOk1M1S and siXOl1M1S of the present disclosure in order. Furthermore, the inhibition rate of XO mRNA for each siRNA is summarized in Table 9. For siRNAs of the same test group, the XO mRNA inhibition rate is the arithmetic mean of the XO mRNA inhibition rates of the test group measured in three culture wells. In FIG. 2, siRNAs 7 to 12 corresponded to siXOg1M1S, siXOh1M1S, siXOi1M1S, siXOj1M1S, siXOk1M1S and siXOl1M1S in order.

[0567] [Table 9]

[0568] As can be seen from the results in Table 9, the siRNAs provided by the present disclosure exhibited high XO mRNA suppression activity in primary mouse liver cells, and at an siRNA concentration of 20 nM, the XO mRNA suppression rate was at least 78.95%, and even reached 88.07%.

[0569] Experimental Example 4 Suppression of XO mRNA expression by siRNA complex in mice C57BL / 6N mice were randomly divided into groups of 5 mice (all females) and numbered. Each group was subcutaneously injected with siRNA complexes L10-siXOi1M1S, L10-siXOk1M1S, and comparative siRNA complex NC at a dose of 3 mg / kg (as siRNA). Each siRNA complex was provided as a 0.9% sodium chloride solution containing 0.6 mg / ml (as siRNA) of siRNA complex, and the administration volume was 5 ml / kg for each group.

[0570] As a blank control group, one of the mice was administered 1×PBS at a volume of 5 ml / kg.

[0571] The animals were sacrificed 7 days after administration, and the liver tissue of each mouse was collected and preserved in RNA later (Sigma Aldrich). The liver tissue was homogenized using a tissue homogenizer, and further extracted with Trizol (Thermo Fisher) according to the operating steps described in the manufacturer's instructions to obtain total RNA.

[0572] ImProm-II TMThe total RNA extracted was reverse transcribed into cDNA using a reverse transcription kit (Promega) according to the instructions to obtain a solution containing cDNA, and then the expression level of XO mRNA in liver tissue was detected using a fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.). Fluorescent quantitative PCR detection and calculation of the expression level and inhibition rate of XO mRNA were performed according to the method of Experimental Example 3. In the fluorescent quantitative PCR method, mouse GAPDH (mGAPDH) gene was used as the endogenous reference gene, and primers for XO and primers for mouse GAPDH were used to detect XO and mouse GAPDH, respectively. The sequences of the detection primers are shown in Table 8. The XO mRNA expression level of the blank control group was set to 100%, and the inhibition rate of the XO mRNA expression level was set to 0% accordingly. The test results of the test group administered with the siRNA complex were standardized by the XO mRNA expression level of the control group, and the results are shown in Figure 3 and Table 10. In FIG. 3, complex 1 was L10-siXOi1M1S and complex 2 was L10-siXOk1M1S.

[0573] [Table 10]

[0574] As can be seen from the results in Table 10, the siRNA complex of the present disclosure exhibited an excellent XO mRNA suppression effect, with an XO mRNA suppression rate of at least 70.9%, and even as high as 76.2%, at an siRNA concentration of 3 mg / kg.

[0575] Although several embodiments of the present disclosure have been described in detail above, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical idea of ​​the present disclosure, and all of these simple modifications fall within the scope of protection of the present disclosure.

[0576] It should also be noted that the specific technical features described in the above embodiments may be combined in any suitable manner if there is no contradiction, and in order to avoid unnecessary duplication, the present disclosure does not separately describe various possible combination methods.

[0577] Furthermore, various different embodiments of the present disclosure can be arbitrarily combined, and such combinations should also be considered as being disclosed in the present disclosure, unless they deviate from the spirit of the present disclosure.

Claims

1. An siRNA complex that inhibits expression of the xanthine oxidase (XO) gene in hepatocytes, comprising: The siRNA conjugate has the structure shown in formula (308): 【Chemistry 1】 During the ceremony, n1 is an integer selected from 1 to 2, n3 is an integer selected from 0 to 1, and n1+n3=2 to 3; Each m1, m2, or m3 is independently an integer selected from 2 to 10; each R 10 , R 11 , R 12 , R 13 , R 14 or R 15 is independently selected from the group consisting of H, a methyl group and an ethyl group; R 3 is a group having a structure represented by formula (A59), 【Chemistry 2】 In the formula, E 1 is OH, SH or BH 2 ; Nu is siRNA, the siRNA comprises a sense strand and an antisense strand, each nucleotide of the siRNA being independently modified or unmodified; the sense strand and the antisense strand have the same or different lengths; the sense strand has a length of 19-23 nucleotides and the antisense strand has a length of 19-26 nucleotides; The sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II form a double-stranded region that is substantially reverse-complementary or completely reverse-complementary, and the nucleotide sequence I and the nucleotide sequence II are one selected from the sequences shown in i) to xii) below; i) said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length and have no more than one nucleotide difference, and said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 are equal in length and have no more than one nucleotide difference; 5'-GAGAUGAAGUUCAAGAAUZ １ -3' (SEQ ID NO:1), 5'-Z ２ AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 2) However, Z １ is A and Z ２ is U, and in said nucleotide sequence I, position Z １ Nucleotide Z corresponding to ３ and in said nucleotide sequence II, position Z ２ Nucleotide Z corresponding to ４ and Z ４ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 is at position Z 4 , where Z 4 is selected from A, C, or G; ii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 61 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 are equal in length and have no more than one nucleotide difference; 5'-CAUAACUGGAAUUUGUAAZ ５ -3' (SEQ ID NO:61), 5'-Z ６ UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:62) However, Z ５ is U and Z ６ is A, and in said nucleotide sequence I, position Z ５ Nucleotide Z corresponding to ７ and in said nucleotide sequence II, position Z ６ Nucleotide Z corresponding to ８ and Z ８ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:62 is at position Z 8 , where Z 8 is selected from U, C, or G; iii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 are equal in length and have no more than one nucleotide difference; 5'-CAUUAUCACAAUUGAGAZ ９ -3' (SEQ ID NO: 121), 5'-Z １０ UCCUCAAUUGUGAUAAUG-3' (SEQ ID NO: 122) However, Z ９ is U and Z １０ is A, and in said nucleotide sequence I, position Z ９ Nucleotide Z corresponding to １１ and in said nucleotide sequence II, position Z １０ Nucleotide Z corresponding to １２ and Z １２ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:122 is at position Z 12 , where Z 12 is selected from U, C, or G; iv) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 are equal in length and have no more than one nucleotide difference; 5'-GGAUCUCUCUCAGAGUAUZ １３ -3' (SEQ ID NO: 181), 5'-Z １４ AUACUCUGAGAGAGAGAUCC-3 '(SEQ ID NO: 182) However, Z １３ is U and Z １４ is A, and in said nucleotide sequence I, position Z １３ Nucleotide Z corresponding to １５ and in said nucleotide sequence II, position Z １４ Nucleotide Z corresponding to １６ and Z １６ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:182 is at position Z 16 , where Z 16 is selected from U, C, or G; v) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 are equal in length and have no more than one nucleotide difference; 5'-ACAAUGGACAACUGCUAUAZ １７ -3' (SEQ ID NO: 241), 5'-Z １８ UAUAGCAGUUGUCCAUGU-3 '(SEQ ID NO: 242) However, Z １７ is A and Z １８ is U, and in said nucleotide sequence I, position Z １７ Nucleotide Z corresponding to １９ and in said nucleotide sequence II, position Z １８ Nucleotide Z corresponding to ２０ and Z ２０ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:242 is at position Z20, where Z20 is selected from A, C, or G; vi) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 302 are equal in length and have no more than one nucleotide difference; 5'-UAGCAAGCUCUCAGUAUCZ ２１ -3' (SEQ ID NO: 301), 5'-Z ２２ GAUACUGAGAGCUUGCUA-3 '(SEQ ID NO: 302) However, Z ２１ is A and Z ２２ is U, and in said nucleotide sequence I, position Z ２１ Nucleotide Z corresponding to ２３ and in said nucleotide sequence II, position Z ２２ Nucleotide Z corresponding to ２４ and Z ２４ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:302 is at position Z24, where Z24 is selected from A, C, or G; vii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 362 are equal in length and have no more than one nucleotide difference; 5'-AUAAGGUUACUUGUGUUGZ ２５ -3' (SEQ ID NO: 361), 5'-Z ２６ CAACACAAGUAACCUUAU-3 '(SEQ ID NO:362) However, Z ２５ is G and Z ２６ is C, and in said nucleotide sequence I, position Z ２５ Nucleotide Z corresponding to ２７ and in said nucleotide sequence II, position Z ２６ Nucleotide Z corresponding to ２８ and Z ２８ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:362 is at position Z28, where Z28 is selected from A, U, or G; viii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 422 are equal in length and have no more than one nucleotide difference; 5'-GAAAAUCACCUAUGAAGAZ ２９ -3' (SEQ ID NO:421), 5'-Z ３０ UCUUCAUAGGUGAUUUUC-3 '(SEQ ID NO:422) However, Z ２９ is A and Z ３０ is U, and in said nucleotide sequence I, position Z ２９ Nucleotide Z corresponding to ３１ and in said nucleotide sequence II, position Z ３０ Nucleotide Z corresponding to ３２ and Z ３２ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:422 is at position Z 32 , where Z 32 is selected from A, C, or G; ix) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 481 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 482 are equal in length and have no more than one nucleotide difference; 5'-GAUGCUAUAAAGAACAACZ ３３ -3' (SEQ ID NO:481), 5'-Z ３４ GUUGUUCUUUAUAGCAUC-3 '(SEQ ID NO:482) However, Z ３３ is U and Z ３４ is A, and in said nucleotide sequence I, position Z ３３ Nucleotide Z corresponding to ３５ and in said nucleotide sequence II, position Z ３４ Nucleotide Z corresponding to ３６ and Z ３６ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:482 is at position Z 36 , where Z 36 is selected from U, C, or G; x) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 541 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 542 are equal in length and have no more than one nucleotide difference; 5'-GAACAACUCCUUUUAUGGZ ３７ -3' (SEQ ID NO:541), 5'-Z ３８ CCAUAAAAGGAGUUGUUC-3' (SEQ ID NO:542) However, Z ３７ is A and Z ３８ is U, and in said nucleotide sequence I, position Z ３７ Nucleotide Z corresponding to ３９ and in said nucleotide sequence II, position Z ３８ Nucleotide Z corresponding to ４０ and Z ４０ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:542 is at position Z 40 , where Z 40 is selected from A, C, or G; xi) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 601 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 602 are equal in length and have no more than one nucleotide difference; 5'-CUUGCUCUGAAGUAGAAAZ ４１ -3' (SEQ ID NO:601), 5'-Z ４２ AUUUCUACUUCAGAGCAAG-3 '(SEQ ID NO:602) However, Z ４１ is U and Z ４２ is A, and in said nucleotide sequence I, position Z ４１ Nucleotide Z corresponding to ４３ and in said nucleotide sequence II, position Z ４２ Nucleotide Z corresponding to ４４ and Z ４４ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:602 is at position Z 44 , where Z 44 is selected from U, C, or G; xii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 661 are equal in length and have no more than one nucleotide difference, and the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 662 are equal in length and have no more than one nucleotide difference; 5'-CUUCUUUGCCAUCAAAGAZ ４５ -3' (SEQ ID NO:661), 5'-Z ４６ UCUUUGAUGGCAAAAGAAG-3' (SEQ ID NO:662) However, Z ４５ is U and Z ４６ is A, and in said nucleotide sequence I, position Z ４５ Nucleotide Z corresponding to ４７ and in said nucleotide sequence II, position Z ４６ Nucleotide Z corresponding to ４８ and Z ４８ is the first nucleotide at the 5' end of the antisense strand, and the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO:662 is at position Z 48 , where Z 48 is selected from U, C, or G; R 2 is a straight chain alkylene group of from 1 to 20 carbon atoms in length, where one or more of the carbon atoms are optionally substituted with one or more selected from the group consisting of C(O), NH, O, S, CH═N, S(O) 2 , a C 2 -C 10 alkenylene group, a C 2 -C 10 alkynylene group, a C 6 -C 10 arylene group, a C 3 -C 18 heterocyclylene group, and a C 5 -C 10 heteroarylene group; R 2 is a C 1 -C 10 alkyl group, a C 6 -C 10 aryl group, a C 5 -C 10 heteroaryl group, a C 1 -C 10 halogenated alkyl group, a —OC 1 -C 10 alkyl group, a —OC 1 -C 10 alkylphenyl group, a —C 1 -C 10 Alkyl-OH, -OC 1 -C 10 halogenated alkyl group, -SC 1 -C 10 alkyl group, -SC 1 -C 10 alkylphenyl group, -C 1 -C 10 alkyl-SH, -SC 1 -C 10 halogenated alkyl group, halogen substituent, -OH, -SH, -NH 2 , -C 1 -C 10 alkyl-NH 2 , -N(C 1 -C 10 alkyl group)(C 1 -C 10 alkyl group), -NH(C 1 -C 10 alkyl group), -N(C 1 -C 10 alkyl group)(C 1 -C 10 alkylphenyl group), -NH(C 1 -C 10 alkylphenyl group), cyano group, nitro group, -CO 2 H, -C(O)O(C 1 -C 10 alkyl group), -CON(C 1-C 10 alkyl group) (C 1 -C 10 alkyl group), -CONH(C 1 -C 10 alkyl group), -CONH 2 , -NHC(O)(C 1 -C 10 alkyl group), -NHC(O)(phenyl group), -N(C 1 -C 10 alkyl group)C(O)(C 1 -C 10 alkyl group), -N(C 1 -C 10 alkyl group)C(O)(phenyl group), -C(O)C 1 -C 10 alkyl group, -C(O)C 1 -C 10 alkylphenyl group, -C(O)C 1 -C 10 haloalkyl group, -OC(O)C 1 -C 10 alkyl group, -SO 2 (C 1 -C 10 alkyl group), -SO 2 (phenyl group), -SO 2 (C 1 -C 10 Halogenated alkyl groups), -SO 2 NH 2 , -SO 2may optionally have one or more substituents selected from the group consisting of NH (C 1 -C 10 alkyl group), -SO 2 NH (phenyl group), -NHSO 2 (C 1 -C 10 alkyl group), -NHSO 2 (phenyl group) and -NHSO 2 (C 1 -C 10 halogenated alkyl group); each L 1 is independently a straight chain alkylene of 1 to 70 carbon atoms in length, where one or more of the carbon atoms is optionally substituted with one or more selected from the group consisting of C(O), NH, O, S, CH═N, S(O) 2 , a C 2 -C 10 alkenylene group, a C 2 -C 10 alkynylene group, a C 6 -C 10 arylene group, a C 3 -C 18 heterocyclylene group, and a C 5 -C 10 heteroarylene group; L 1 is a C 1 -C 10 alkyl group, a C 6 -C 10 aryl group, a C 5 -C 10 heteroaryl group, a C 1 -C 10 halogenated alkyl group, a —OC 1 -C 10 alkyl group, a —OC 1 -C 10 alkylphenyl group, a —C 1 -C 10 Alkyl-OH, -OC 1 -C 10 halogenated alkyl group, -SC 1 -C 10 alkyl group, -SC 1 -C 10 alkylphenyl group, -C 1 -C 10 alkyl-SH, -SC 1 -C 10 halogenated alkyl group, halogen substituent, -OH, -SH, -NH 2 , -C 1 -C 10 alkyl-NH 2 , -N(C 1 -C 10 alkyl group)(C 1 -C 10 alkyl group), -NH(C 1 -C 10 alkyl group), -N(C 1 -C 10 alkyl group)(C 1 -C 10 alkylphenyl group), -NH(C 1 -C 10 alkylphenyl group), cyano group, nitro group, -CO 2 H, -C(O)O(C 1 -C 10 alkyl group), -CON(C 1-C 10 alkyl group) (C 1 -C 10 alkyl group), -CONH(C 1 -C 10 alkyl group), -CONH 2 , -NHC(O)(C 1 -C 10 alkyl group), -NHC(O)(phenyl group), -N(C 1 -C 10 alkyl group)C(O)(C 1 -C 10 alkyl group), -N(C 1 -C 10 alkyl group)C(O)(phenyl group), -C(O)C 1 -C 10 alkyl group, -C(O)C 1 -C 10 alkylphenyl group, -C(O)C 1 -C 10 haloalkyl group, -OC(O)C 1 -C 10 alkyl group, -SO 2 (C 1 -C 10 alkyl group), -SO 2 (phenyl group), -SO 2 (C 1 -C 10 Halogenated alkyl groups), -SO 2 NH 2 , -SO2NH (C 1 -C 10 alkyl group), -SO 2 NH (phenyl group), -NHSO 2 (C 1 -C 10 alkyl group), -NHSO 2 (phenyl group) and -NHSO 2 (C 1 -C 10 halogenated alkyl group), 【change】 represents the site at which the group is covalently attached, M 1 represents a targeting group in the siRNA conjugate.

2. The nucleotide sequence I is a nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence II is a nucleotide sequence shown in SEQ ID NO: 4, 5'-GAGAUGAAGUUCAAGAAUZ ３ -3' (SEQ ID NO:3), 5'-Z ４ AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 4) However, Z ４ is selected from A, U, G or C; Z ３ Is, Z ４ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:63, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:64; 5'-CAUAACUGGAAUUUGUAAZ ７ -3' (SEQ ID NO:63), 5'-Z ８ UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:64) However, Z ８ is selected from A, U, G or C; Z ７ Is, Z ８ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 123, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124; 5'-CAUUAUCACAAUUGAGAZ １１ -3' (SEQ ID NO: 123), 5'-Z １２ UCCUCAAUUGUGAUAAUG-3' (SEQ ID NO: 124) However, Z １２ is selected from A, U, G or C; Z １１ Is, Z １２ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 183, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 184; 5'-GGAUCUCUCUCAGAGUAUZ １５ -3' (SEQ ID NO: 183), 5'-Z １６ AUACUCUGAGAGAGAGAUCC-3 '(SEQ ID NO: 184) However, Z １６ is selected from A, U, G or C; Z １５ Is, Z １６ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 243, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 244; 5'-ACAAUGGACAACUGCUAUAZ １９ -3' (SEQ ID NO: 243), 5'-Z ２０ UAUAGCAGUUGUCCAUGU-3 '(SEQ ID NO: 244) However, Z ２０ is selected from A, U, G or C; Z １９ Is, Z ２０ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 304; 5'-UAGCAAGCUCUCAGUAUCZ ２３ -3' (SEQ ID NO: 303), 5'-Z ２４ GAUACUGAGAGCUUGCUA-3 '(SEQ ID NO: 304) However, Z ２４ is selected from A, U, G or C; Z ２３ Is, Z ２４ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 363, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 364; 5'-AUAAGGUUACUUGUGUUGZ ２７ -3' (SEQ ID NO: 363), 5'-Z ２８ CAACACAAGUAACCUUAU-3 '(SEQ ID NO: 364) However, Z ２８ is selected from A, U, G or C; Z ２７ Is, Z ２８ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 423, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 424; 5'-GAAAAUCACCUAUGAAGAZ ３１ -3' (SEQ ID NO:423), 5'-Z ３２ UCUUCAUAGGUGAUUUUC-3 '(SEQ ID NO:424) However, Z ３２ is selected from A, U, G or C; Z ３１ Is, Z ３２ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 483, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 484; 5'-GAUGCUAUAAAGAACAACZ ３５ -3' (SEQ ID NO:483), 5'-Z ３６ GUUGUUCUUUAUAGCAUC-3 '(SEQ ID NO:484) However, Z ３６ is selected from A, U, G or C; Z ３５ Is, Z ３６ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:543, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO:544; 5'-GAACAACUCCUUUUAUGGZ ３９ -3' (SEQ ID NO:543), 5'-Z ４０ CCAUAAAAGGAGUUGUUC-3' (SEQ ID NO:544) However, Z ４０ is selected from A, U, G or C; Z ３９ Is, Z ４０ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 603, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 604; 5'-CUUGCUCUGAAGUAGAAAZ ４３ -3' (SEQ ID NO:603), 5'-Z ４４ UUUCUACUUCAGAGCAAG-3 '(SEQ ID NO:604) However, Z ４４ is selected from A, U, G or C; Z ４３ Is, Z ４４ is a nucleotide complementary to Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 663, and the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 664; 5'-CUUCUUUGCCAUCAAAGAZ ４７ -3' (SEQ ID NO:663), 5'-Z ４８ UCUUUGAUGGCAAAAGAAG-3' (SEQ ID NO:664) However, Z ４８ is selected from A, U, G or C; Z ４７ Is, Z ４８ The siRNA complex of claim 1 , wherein the nucleotide is complementary to:

3. 3. The siRNA complex of claim 1, wherein the sense strand further comprises a nucleotide sequence III and the antisense strand further comprises a nucleotide sequence IV, wherein the length of nucleotide sequence III and the length of nucleotide sequence IV are each independently 1 to 4 nucleotides, wherein the nucleotide sequence III is linked to the 5' end of the nucleotide sequence I and the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II, wherein the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary, wherein the substantially reverse complementary refers to the presence of not more than one base mismatch between the two nucleotide sequences and the completely reverse complementary refers to the absence of mismatch between the two nucleotide sequences.

4. the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:1, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is U; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is UU; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is AUU; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is CAUU; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:61, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is A; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III is AA from the 5' to the 3' end; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III is UAA from the 5' to the 3' end; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III is GUAA from the 5' to the 3' end; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 121, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is C; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III is GC from the 5' to the 3' end; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III is AGC from the 5' to the 3' end; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III is CAGC from the 5' to the 3' end; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 181, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is A; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is CA; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is CCA; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is CCCA; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:241, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is C; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is CC; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is UCC; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is UUCC; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 301, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is C; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is CC; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is GCC; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is UGCC; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 361, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is G; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is GG; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is AGG; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is AAGG; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 421, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is U; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is GU; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is GGU; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III from the 5' to the 3' end is GGGU; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 481, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is G; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III is AG from the 5' to the 3' end; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III is GAG from the 5' to the 3' end; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III is UGAG from the 5' to the 3' end; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:541, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is A; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III is AA from the 5' to the 3' end; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III is AAA from the 5' to the 3' end; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III is UAAA from the 5' to the 3' end; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:601, and the nucleotide sequences III and IV are each 1 nucleotide in length, and the base of the nucleotide sequence III is G; or the nucleotide sequences III and IV are each 2 nucleotides in length, and the base sequence of the nucleotide sequence III is GG from the 5' to the 3' end; or the nucleotide sequences III and IV are each 3 nucleotides in length, and the base sequence of the nucleotide sequence III is UGG from the 5' to the 3' end; or the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of the nucleotide sequence III is GUGG from the 5' to the 3' end; Alternatively, the nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO:661, and the nucleotide sequences III and IV are each 1 nucleotide in length and the base of the nucleotide sequence III is U; or the nucleotide sequences III and IV are each 2 nucleotides in length and the base sequence of the nucleotide sequence III is AU from the 5' to the 3' end; or the nucleotide sequences III and IV are each 3 nucleotides in length and the base sequence of the nucleotide sequence III is UAU from the 5' to the 3' end; or the nucleotide sequences III and IV are each 4 nucleotides in length and the base sequence of the nucleotide sequence III is CUAU from the 5' to the 3' end. The siRNA complex of claim 3.

5. The siRNA complex of any one of claims 1 to 4, wherein the antisense strand further comprises a nucleotide sequence V, the nucleotide sequence V being 1 to 3 nucleotides in length and attached to the 3' end of the antisense strand, constituting a 3' overhang of the antisense strand.

6. The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:5, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:6; 5'-GAGAUGAAGUUCAAGAAUZ ３ -3' (SEQ ID NO:5), 5'-Z ４ AUUCUUGAACUUCAUCUCAA-3' (SEQ ID NO: 6) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 8; 5'-UUGAGAUGAAGUUCAAGAAUZ ３ -3' (SEQ ID NO:7), 5'-Z ４ AUUCUUGAACUUCAUCUCAAUG-3' (SEQ ID NO: 8) However, the Z ４ is the first nucleotide at the 5' end of the antisense strand, and Z ４ is selected from A, U, G or C; Z ３ Is, Z ４ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:65, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:66; 5'-CAUAACUGGAAUUUGUAAZ ７ -3' (SEQ ID NO:65), 5'-Z ８ UUACAAAUUCCAGUUAUGUU-3' (SEQ ID NO:66) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:67, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:68; 5'-AACAUAACUGGAAUUUGUAAZ ７ -3' (SEQ ID NO:67), 5'-Z ８ UUACAAAUUCCAGUUAUGUUAC-3' (SEQ ID NO:68) However, the Z ８ is the first nucleotide at the 5' end of the antisense strand, and Z ８ is selected from A, U, G or C; Z ７ Is, Z ８ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 125, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 126; 5'-CAUUAUCACAAUUGAGAZ １１ -3' (SEQ ID NO: 125), 5'-Z １２ UCCUCAAUUGUGAUAAUGGC-3 '(SEQ ID NO: 126) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 127, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 128; 5'-GCCAUUAUCACAAUUGAGGAZ １１ -3' (SEQ ID NO: 127), 5'-Z １２ UCCUCAAUUGUGAUAAUGGCUG-3 '(SEQ ID NO: 128) However, the Z １２ is the first nucleotide at the 5' end of the antisense strand, and Z １２ is selected from A, U, G or C; Z １１ Is, Z １２ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 185, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 186; 5'-GGAUCUCUCUCAGAGUAUZ １５ -3' (SEQ ID NO: 185), 5'-Z １６ AUACUCUGAGAGAGAUCCUG-3 '(SEQ ID NO: 186) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 187, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 188; 5'-CAGGAUCUCUCUCAGAGUAUZ １５ -3' (SEQ ID NO: 187), 5'-Z １６ AUACUCUGAGAGAGAUCCUGGG-3 '(SEQ ID NO: 188) However, the Z １６ is the first nucleotide at the 5' end of the antisense strand, and Z １６ is selected from A, U, G or C; Z １５ Is, Z １６ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 245, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 246; 5'-ACAAUGGACAACUGCUAUAZ １９ -3' (SEQ ID NO: 245), 5'-Z ２０ UAUAGCAGUUGUCCAUGUGGG-3 '(SEQ ID NO:246) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 247, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 248; 5'-CCACAUGGACAACUGCUAUAZ １９ -3' (SEQ ID NO: 247), 5'-Z ２０ UAUAGCAGUUGUCCAUGUGGAA-3 '(SEQ ID NO: 248) wherein Z20 is the first nucleotide at the 5' end of the antisense strand, and Z ２０ is selected from A, U, G or C; Z １９ Is, Z ２０ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 305, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 306; 5'-UAGCAAGCUCUCAGUAUCZ ２３ -3' (SEQ ID NO: 305), 5'-Z ２４ GAUACUGAGAGCUUGCUAGG-3 '(SEQ ID NO: 306) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 307, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 308; 5'-CCUAGCAAGCUCUCAGUAUCZ ２３ -3' (SEQ ID NO: 307), 5'-Z ２４ GAUACUGAGAGCUUGCUAGGCA-3 '(SEQ ID NO: 308) However, the Z ２４ is the first nucleotide at the 5' end of the antisense strand, and Z ２４ is selected from A, U, G or C; Z ２３ Is, Z ２４ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 365, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 366; 5'-AUAAGGUUACUUGUGUUGZ ２７ -3' (SEQ ID NO: 365), 5'-Z ２８ CAACACAAGUAACCUUAUCC-3 '(SEQ ID NO: 366) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 367, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 368; 5'-GGAUAAGGUUACUUGUGUUGZ ２７ -3' (SEQ ID NO: 367), 5'-Z ２８ CAACACAAGUAACCUUAUCCUU-3 '(SEQ ID NO: 368) However, the Z ２８ is the first nucleotide at the 5' end of the antisense strand, and Z ２８ is selected from A, U, G or C; Z ２７ Is, Z ２８ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 425, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 426; 5'-GAAAAUCACCUAUGAAGAZ ３１ -3' (SEQ ID NO:425), 5'-Z ３２ UCUUCAUAGGUGAUUUUCAC-3 '(SEQ ID NO:426) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 427, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 428; 5'-GUGAAAAUCACCUAUGAAGAZ ３１ -3' (SEQ ID NO:427), 5'-Z ３２ UCUUCAUAGGUGAUUUUCACCC-3 '(SEQ ID NO:428) However, the Z ３２ is the first nucleotide at the 5' end of the antisense strand, and Z ３２ is selected from A, U, G or C; Z ３１ Is, Z ３２ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 485, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 486; 5'-GAUGCUAUAAAGAACAACZ ３５ -3' (SEQ ID NO:485), 5'-Z ３６ GUUGUUCUUUAUAGCAUCCU-3 '(SEQ ID NO:486) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 487, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 488; 5'-AGGAUGCUAUAAAGAACAACZ ３５ -3' (SEQ ID NO:487), 5'-Z ３６ GUUGUUCUUUAUAGCAUCCUCA-3 '(SEQ ID NO:488) However, the Z ３６ is the first nucleotide at the 5' end of the antisense strand, and Z ３６ is selected from A, U, G or C; Z ３５ Is, Z ３６ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:545, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:546; 5'-GAACAACUCCUUUUAUGGZ ３９ -3' (SEQ ID NO:545), 5'-Z ４０ CCAUAAAAGGAGUUGUUCUU-3 '(SEQ ID NO:546) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:547, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:548; 5'-AAGAACAACUCCUUUUAUGGZ ３９ -3' (SEQ ID NO:547), 5'-Z ４０ CCAUAAAAGGAGUUGUUCUUUA-3 '(SEQ ID NO:548) However, the Z ４０ is the first nucleotide at the 5' end of the antisense strand, and Z ４０ is selected from A, U, G or C; Z ３９ Is, Z ４０ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:605, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:606; 5'-CUUGCUCUGAAGUAGAAAZ ４３ -3' (SEQ ID NO:605), 5'-Z ４４ UUUCUACUUCAGAGCAAGCC-3 '(SEQ ID NO:606) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 607, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 608; 5'-GGCUUGCUCUGAAGUAGAAAZ ４３ -3' (SEQ ID NO:607), 5'-Z ４４ UUUCUACUUCAGAGCAAGCCAC-3 '(SEQ ID NO:608) However, the Z ４４ is the first nucleotide at the 5' end of the antisense strand, and Z ４４ is selected from A, U, G or C; Z ４３ Is, Z ４４ is a nucleotide complementary to Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:665, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:666; 5'-CUUCUUUGCCAUCAAAGAZ ４７ -3' (SEQ ID NO:665), 5'-Z ４８ UCUUUGAUGGGCAAAGAAGAU-3 '(SEQ ID NO:666) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:667, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:668; 5'-AUCUUCUUUGCCAUCAAAGAZ ４７ -3' (SEQ ID NO:667), 5'-Z ４８ UCUUUGAUGGCAAAAGAAGAUAG-3' (SEQ ID NO:668) However, the Z ４８ is the first nucleotide at the 5' end of the antisense strand, and Z ４８ is selected from A, U, G or C; Z ４７ Is, Z ４８ The siRNA complex according to any one of claims 1 to 5, which is a nucleotide complementary to:

7. each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide; The fluoro-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and from the 5' to the 3' end, at least the 7th, 8th, and 9th nucleotides of nucleotide sequence I are fluoro-modified nucleotides, and from the 5' to the 3' end, at least the 2nd, 6th, 14th, and 16th nucleotides of nucleotide sequence II are fluoro-modified nucleotides. The siRNA complex of any one of claims 1 to 6.

8. The siRNA complex of claim 7, wherein each of the non-fluoro-modified nucleotides is a methoxy-modified nucleotide, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxy group of the ribose group is substituted with methoxy.

9. From the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides; from the 5' to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides; Alternatively, from the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides; and from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides; Alternatively, from the 5' to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides. The siRNA complex of claim 8.

10. In the siRNA, at least one phosphate group is a thiophosphate group, the thiophosphate group being Between the first and second nucleotides from the 5' end of the sense strand, Between the second and third nucleotides from the 5' end of the sense strand, Between the first and second nucleotides from the 3' end of the sense strand, Between the second and third nucleotides from the 3' end of the sense strand, Between the first and second nucleotides from the 5' end of the antisense strand, Between the second and third nucleotides from the 5' end of the antisense strand, Between the first and second nucleotides from the 3' end of the antisense strand, and The siRNA complex according to any one of claims 1 to 9, wherein the siRNA complex is bound to at least one of the groups consisting of the second nucleotide and the third nucleotide from the 3' end of the antisense strand.

11. The 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide, Alternatively, the 5'-phosphate nucleotide is a nucleotide having a structure represented by formula (2), and the 5'-phosphate analog modified nucleotide is selected from nucleotides having a structure represented by any one of formulas (3) to (6): 【Chemistry 3】 In the formula, R is selected from H, OH, a methoxy group, or fluorine, and Base represents a base selected from A, U, C, G, or T. The siRNA complex according to claim 1 ,

12. The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 9, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 10; Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 11, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 12; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 69, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 70; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 71, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 72; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 129, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 130; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 131, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 132; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 189, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 190; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 191, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 192; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 249, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 250; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 251, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 252; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 309, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 310; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 311, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 312; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 369, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 370; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 371, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 372; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 429, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 430; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 431, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 432; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 489, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 490; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 491, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 492; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:549, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:550; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 551, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 552; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 609, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 610; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:611, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:612; or The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 13, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 14; Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 15, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 16; Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 17, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 18; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 19, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 20; Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 21, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 22; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 23, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 24; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 73, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 74; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 75, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 76; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 77, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 78; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 79, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 80; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 81, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 82; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 83, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 84; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 133, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 134; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 135, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 136; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 137, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 138; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 139, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 140; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 141, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 142; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 143, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 144; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 193, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 194; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 195, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 196; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 197, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 198; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 199, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 200; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 201, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 202; Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 203, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:

204. Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 253, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 254; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 255, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 256; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 257, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 258; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 259, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 260; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 261, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 262; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 263, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 264; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 313, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 314; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 315, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 316; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 317, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 318; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 319, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 320; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 321, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 322; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 323, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 324; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 373, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 374; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 375, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 376; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 377, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 378; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 379, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 380; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 381, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 382; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 383, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 384; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 433, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 434; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 435, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 436; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 437, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 438; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 439, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 440; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 441, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 442; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 443, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 444; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 493, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 494; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 495, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 496; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 497, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 498; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 499, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 500; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 501, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 502; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 503, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 504; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 553, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 554; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 555, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 556; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:557, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:558; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 559, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 560; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 561, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 562; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 563, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 564; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 613, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 614; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 615, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 616; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 617, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 618; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 619, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 620; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 621, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 622; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 623, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 624; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 673, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 674; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 675, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 676; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 677, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 678; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 679, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 680; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 681, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 682; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 683, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 684; or The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:25, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:26; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 27, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 28; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 29, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 30; Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 31, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 32; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 33, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 34; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 35, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 36; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 85, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 86; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 87, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 88; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 89, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 90; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 91, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 92; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 93, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 94; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 95, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 96; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 145, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 146; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 147, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 148; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 149, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 150; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 151, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 152; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 153, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 154; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 155, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 156; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 205, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 206; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 207, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 208; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 209, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 210; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 211, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 212; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 213, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 214; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 215, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 216; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 265, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 266; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 267, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 268; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 269, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 270; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 271, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 272; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 273, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 274; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 275, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 276; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 325, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 326; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 327, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 328; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 329, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 330; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 331, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 332; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 333, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 334; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 335, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 336; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 385, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 386; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 387, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 388; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 389, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 390; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 391, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 392; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 393, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 394; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 395, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 396; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 445, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 446; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 447, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 448; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 449, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 450; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 451, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 452; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 453, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 454; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 455, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 456; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:505, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:506; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 507, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 508; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 509, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 510; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:511, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:512; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:513, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:514; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:515, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:516; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:565, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:566; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:567, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:568; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:569, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:570; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:571, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:572; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:573, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:574; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:575, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:576; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 625, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 626; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 627, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 628; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 629, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 630; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 631, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 632; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 633, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 634; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 635, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 636; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 685, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 686; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 687, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 688; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 689, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 690; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 691, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 692; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 693, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 694; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:695, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:696; or The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 37, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 38; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 39, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 40; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 41, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 42; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 43, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 44; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 45, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 46; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 47, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 48; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 49, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 50; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:51, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:52; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:53, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:54; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:55, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:56; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:57, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:58; Or, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:59, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:60; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 97, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 98; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 99, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 100; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 101, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 102; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 103, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 104; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 105, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 106; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 107, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 108; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 109, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 110; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 111, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 112; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 113, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 114; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 115, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 116; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 117, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 118; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 119, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 120; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 157, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 158; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 159, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 160; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 161, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 162; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 163, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 164; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 165, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 166; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 167, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 168; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 169, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 170; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 171, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 172; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 173, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 174; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 175, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 176; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 177, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 178; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 179, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 180; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 217, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 218; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 219, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 220; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 221, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 222; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 223, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 224; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 225, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 226; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 227, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 228; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 229, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 230; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 231, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 232; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 233, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 234; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 235, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 236; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 237, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 238; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 239, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 240; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 277, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 278; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 279, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 280; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 281, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 282; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 283, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 284; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 285, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 286; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 287, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 288; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 289, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 290; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 291, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 292; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 293, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 294; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:295, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:296; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:297, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:298; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 299, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 300; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 337, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 338; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 339, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 340; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 341, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 342; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 343, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 344; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 345, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 346; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 347, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 348; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 349, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 350; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 351, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 352; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 353, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 354; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 355, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 356; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 357, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 358; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 359, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 360; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 397, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 398; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 399, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 400; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 401, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 402; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 403, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 404; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 405, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 406; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 407, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 408; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 409, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 410; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 411, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 412; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 413, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 414; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 415, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 416; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 417, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 418; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 419, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 420; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 457, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 458; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 459, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 460; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 461, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 462; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 463, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 464; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 465, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 466; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 467, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 468; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 469, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 470; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 471, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 472; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 473, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 474; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 475, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 476; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 477, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 478; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 479, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 480; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:517, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:518; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 519, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 520; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 521, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 522; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 523, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 524; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:525, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:526; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:527, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:528; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 529, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 530; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:531, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:532; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 533, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 534; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:535, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:536; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:537, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:538; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 539, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 540; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:577, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:578; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:579, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:580; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 581, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 582; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 583, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 584; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:585, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:586; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:587, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:588; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:589, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:590; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:591, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:592; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:593, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:594; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:595, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:596; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:597, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:598; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:599, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:600; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 637, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 638; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 639, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 640; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 641, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 642; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 643, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 644; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 645, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 646; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 647, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 648; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 649, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 650; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 651, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 652; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 653, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 654; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 655, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 656; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 657, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 658; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 659, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 660; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 697, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 698; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 699, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 700; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 701, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 702; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 703, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 704; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 705, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 706; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 707, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 708; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 709, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 710; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 711, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 712; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 713, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 714; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 715, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 716; Or, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 717, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 718; Alternatively, the siRNA complex of any one of claims 1 to 11, wherein the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 719 and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:

720.

13. Each L １ is independently selected from the group consisting of groups A1-A26, and any combination thereof; 【Chemistry 4】 wherein each j1 is independently an integer from 1 to 20; each j2 is independently an integer from 1 to 20; Each R' is independently C １ -C １０ is an alkyl group, each Ra is selected from the group consisting of A27-A45, and any combination thereof; 【Chemistry 5】 Each Rb is independently C １ -C １０ is an alkyl group, 【change】 The siRNA conjugate of any one of claims 1 to 12, wherein represents the site at which the group is attached to the covalent linking moiety.

14. L １ is selected from the group consisting of groups A1, A4, A5, A6, A8, A10, A11, A13 and combinations thereof; or L １ The siRNA conjugate of claim 13, wherein is a combination of bonds of at least two of groups A1, A4, A8, A10 and A11.

15. L １ The siRNA conjugate of any one of claims 1 to 14, wherein the length of said siRNA conjugate is 3 to 25 atoms.

16. The siRNA conjugate of claim 15, wherein L 1 is 4 to 15 atoms in length.

17. The siRNA complex of any one of claims 1 to 16, wherein each of m1, m2 and m3 is independently an integer from 2 to 5, or m1 = m2 = m3.

18. 18. The siRNA conjugate of any one of claims 1 to 17, wherein each said targeting group is independently a ligand with affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, or wherein at least one of the or each targeting group is galactose or N-acetylgalactosamine.

19. The siRNA complex according to any one of claims 1 to 18, wherein the siRNA complex has a structure as shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422). 【Chemistry 6】 【change】 【change】 【change】 【change】 【change】 【change】

20. The siRNA conjugate of any one of claims 1 to 19, wherein the P atom in formula A59 is attached to the 3' end of the sense strand of the siRNA.

21. A composition for treating and / or preventing uric acid metabolic abnormality or a disease or physiological condition caused thereby, comprising the siRNA complex described in any one of claims 1 to 20.

22. The composition according to claim 21 , wherein the disease or physiological condition caused by abnormal uric acid metabolism is hyperuricemia or gout.

23. A composition for suppressing expression of the XO gene in liver cells, comprising an siRNA complex described in any one of claims 1 to 20.

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