Nucleic acid, pharmaceutical composition, conjugate, preparation method, and use

Abstract

To provide an siRNA that inhibits xanthine oxidase (XO) gene expression, so that diseases caused by abnormal uric acid metabolism, especially hyperuricemia and gout, can be prevented and treated at the cellular level.SOLUTION: The siRNA contains a sense strand and an antisense strand. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide. Lengths of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides. The sense strand contains a specific nucleotide sequence I. The antisense strand contains a specific nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II form a substantially reverse complementary or completely reverse complementary double-stranded region. The siRNA inhibits expression of an XO gene in hepatocytes.SELECTED DRAWING: Figure 1C

Description

[Technical field]

[0001] The present disclosure relates to nucleic acids capable of inhibiting xanthine oxidase (XO) gene expression, including nucleic acids The present disclosure further relates to these nucleic acids, drug compositions and siRNA complexes. The present invention relates to a method for preparing and using the siRNA complex. [Background technology]

[0002] Gout is a disease that is directly related to hyperuricemia due to abnormalities in 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 After the Second World War, the prevalence of the disease has been increasing year by year around the world as countries have developed economically. The number of gout sufferers in China is currently at 12 million.

[0003] Xanthine oxidase (XO) is one of the key targets for treating gout. By suppressing XO expression, the production of hypoxanthine and guanine is effectively suppressed. Furthermore, it is also possible to reduce the production of uric acid, slow down the progression of gout, and improve the condition. By suppressing the expression of the XO gene, diseases caused by abnormalities in uric acid metabolism at the cellular level, In particular, it can prevent and treat hyperuricemia and gout. l interfering RNA (siRNA) is a type of RNA interference (RNA interference). Based on a mechanism called RNA interference (RNAi), The expression of the gene can be inhibited or blocked in a sequence-specific manner to achieve the purpose of treating disease.

[0004] To develop siRNA drugs that suppress XO gene expression and treat diseases caused by abnormal uric acid metabolism In this study, 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 provide the following siRNAs and modified sequences thereof: By having the above structure, the expression of the XO gene can be specifically suppressed, and the drug composition or si The RNA complex can specifically target the liver and suppress the expression of XO genes in the liver. It has been unexpectedly discovered that the present invention can treat or prevent diseases caused by abnormal uric acid metabolism. As a result, the present invention was completed. [Means for solving the problem]

[0006] In some embodiments, the present disclosure provides siRNAs capable of suppressing XO gene expression. The siRNA comprises a sense strand and an antisense strand, and each nucleotide of the siRNA is each nucleotide is independently a modified or unmodified nucleotide, and the sense strand is a nucleotide the antisense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, Sequence I and nucleotide sequence II form at least a part of a double-stranded region in a reverse complementary manner. The nucleotide sequence I and the nucleotide sequence II are shown in the following i) to xii). The sequence is one selected from the sequences listed above.

[0007] i) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length. and the nucleotide difference is three or less, and is shown in nucleotide sequence II and SEQ ID NO:2. The nucleotide sequences to be compared are of equal length and have no more than three nucleotide differences; 5'-GAGAUGAAGUUCAAGAAUZ1-3' (SEQ ID NO: 1), 5'-Z2AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 2) wherein Z1 is A and Z2 is U, and in said nucleotide sequence I, position Z1 is The corresponding nucleotide Z3 is included in the nucleotide sequence II, and the position of Z2 is included in the nucleotide sequence II. and Z4 is the first nucleotide at the 5' end of the antisense strand. It is a nucleotide.

[0008] ii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61 have a length and the nucleotide difference between said nucleotide sequence II and SEQ ID NO:6 is 3 or less. The nucleotide sequence shown in 2 is the same length and has no more than three nucleotide differences. , 5'-CAUAACUGGAAUUUGUAAZ5-3' (SEQ ID NO:61), 5'-Z6UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:62) wherein Z5 is U and Z6 is A; and in said nucleotide sequence I, position Z5 is The corresponding nucleotide Z7 is included in the nucleotide sequence II, which corresponds to Z6 in position II. the antisense strand includes a nucleotide Z8 which is the first nucleotide at the 5' end of the antisense strand; It is a nucleotide.

[0009] iii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 The nucleotide sequence is the same in length and has no more than three nucleotide differences, and is identical to the nucleotide sequence II of SEQ ID NO: The nucleotide sequence shown in No. 122 is the same in length and differs by no more than three nucleotides. and 5'-CAUUAUCACAAUUGAGGAZ9-3' (SEQ ID NO: 121), 5'-Z10 UCCUCAAUUGUGAUAAUG-3' (SEQ ID NO: 122) However, Z9 is U and Z 10 is A, and in said nucleotide sequence I, position Z9 Nucleotide Z corresponding to 11 and in said nucleotide sequence II, position Z 10 To The corresponding nucleotide Z 12 wherein Z 12 is the 5' end of the antisense strand This is the first nucleotide.

[0010] iv) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have a length and the nucleotide sequence II and the sequence of SEQ ID NO: The nucleotide sequence shown in 182 is the same length and has no more than three nucleotide differences. can be, 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 Z1 Nucleotide Z corresponding to 4 16 wherein Z 16 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0011] v) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have a length and the nucleotide difference between said nucleotide sequence II and SEQ ID NO:2 is 3 or less. The nucleotide sequence shown in 42 is the same length and has no more than three nucleotide differences. the law of nature, 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 Z1 Nucleotide Z corresponding to 8 20 wherein Z 20 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0012] vi) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 have the same length and the nucleotide sequence II and the sequence of SEQ ID NO: The nucleotide sequence shown in 302 is the same length and has no more than three nucleotide differences. can be, 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 Z2 Nucleotide Z corresponding to 2 24 wherein Z 24 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0013] vii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 The nucleotide sequence is the same in length and has no more than three nucleotide differences, and is identical to the nucleotide sequence II of SEQ ID NO: The nucleotide sequence shown in No. 362 is the same in length and differs by no more than three nucleotides. and 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 Z2 Nucleotide Z corresponding to 6 28 wherein Z 28 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0014] viii) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421 , which are equal in length and have no more than three nucleotide differences, and which are identical to nucleotide sequence II and sequence The nucleotide sequence shown in reference number 422 is the same length and has no more than three nucleotide differences. Below, 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 Z3 Nucleotide Z corresponds to 0 32 wherein Z 32 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0015] ix) The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 481 have a length and the nucleotide sequence II and the sequence of SEQ ID NO: The nucleotide sequence shown in 482 is the same length and has no more than three nucleotide differences. can be, 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 Z3 Nucleotide Z corresponding to 4 36 wherein Z 36 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0016] x) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 541 have a length and the nucleotide difference between said nucleotide sequence II and SEQ ID NO:5 is 3 or less. The nucleotide sequence shown in 42 is the same length and has no more than three nucleotide differences. the law of nature, 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 Z3 Nucleotide Z corresponding to 8 40 wherein Z 40 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0017] xi) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 601 have a length and the nucleotide sequence II and the sequence of SEQ ID NO: The nucleotide sequence shown in 602 is the same length and has no more than three nucleotide differences. can be, 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 Z4 Nucleotide Z corresponding to 2 44 wherein Z 44 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0018] xii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 661 The nucleotide sequence is the same in length and has no more than three nucleotide differences, and is identical to the nucleotide sequence II of SEQ ID NO: The nucleotide sequence shown in No. 662 is the same in length and differs by no more than three nucleotides. and 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 Z4 Nucleotide Z corresponding to 6 48 wherein Z 48 is the 5' end of the antisense strand It is the first nucleotide at the end.

[0019] In some embodiments, the present disclosure provides a method for the preparation of a siRNA of the present disclosure and a pharma- ceutically acceptable A pharmaceutical composition is provided that includes a carrier.

[0020] In some embodiments, the present disclosure relates to siRNAs and the siRNAs provided by the present disclosure. A siRNA complex is provided that includes a conjugated group that is conjugated and bound to the siRNA.

[0021] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or the siRNA complex, a disease or physiological condition caused by a uric acid metabolic disorder The present invention also provides a use of the compound according to the present invention in the preparation of a medicament for the treatment and / or prophylaxis of

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

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

[0024] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or a kit comprising the siRNA complex. Incorporation by reference

[0025] All publications, patents and patent applications mentioned herein are hereby incorporated by reference in their entirety. Each application is hereby incorporated by reference to the same extent as if each application was specifically and individually incorporated by reference. and is hereby incorporated by reference. Effect of the Invention

[0026] The siRNA, drug composition and siRNA complex provided by the present disclosure have excellent stability. It has high XO mRNA suppression activity, reduced off-target effects, and / or uric acid metabolism. Diseases or physiological conditions caused by abnormalities in uric acid metabolism, especially hyperuricemia and / or gout symptoms. The symptoms can be significantly cured or alleviated.

[0027] In some embodiments, the siRNA, drug composition or s The iRNA complex exhibits excellent target gene silencing activity in in vitro cell experiments. In one embodiment, the siRNA, drug composition, or siRNA complex provided by the present disclosure comprises: At least 20%, 30%, 40%, 50%, 60%, 70%, 80% in hepatocytes, In some embodiments, the present disclosure shows a target gene expression inhibition rate of 90% or 95%. The siRNA provided by It showed high inhibitory activity against the XO target sequence at different siRNA concentrations. showed a certain inhibitory effect, and especially at a concentration of 0.1 nM, the inhibition rate for the target sequence was at least 61.39%, or even as high as 85.43%. The siRNA provided by the disclosure exhibits high suppressive activity in CAL-27 cells, and IC for O mRNA 50 The range is 0.037 to 0.3277 μM. In one embodiment, the siRNA complex provided by the present disclosure is capable of inhibiting the expression of siRNA in mouse primary hepatocytes. It showed high inhibitory activity, and the inhibition rate against XO mRNA was small at a siRNA concentration of 20 nM. It will reach at least 78.95% and even as high as 88.07%.

[0028] In some embodiments, the siRNA, drug composition or s iRNA complexes may have greater stability and / or greater activity in the body. In some embodiments, the siRNA and drug compositions provided by the present disclosure or the siRNA complex is at least 20%, 30%, 40%, 50%, 60%, The target gene expression is suppressed by 70%, 80%, 90% or 95%. In the present disclosure, the siRNA, drug composition, or siRNA complex is At least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or In some embodiments, the present disclosure provides a method for inhibiting the expression of the XO gene by 95%. The siRNA, drug composition or siRNA complex administered is administered to at least 20%, 30%, or more of the body. %, 40%, 50%, 60%, 70%, 80%, 90% or 95% intrahepatic XO gene expression The inhibition rate is shown. In some embodiments, the siRNA, drug, The composition or siRNA complex is administered to the body at least 20%, 30%, 40%, 50%, 60%, Intrahepatic XO gene expression suppression in animal models of 0%, 70%, 80%, 90% or 95% In some embodiments, the siRNA, drug composition provided by the present disclosure The substance or siRNA complex is at least 20%, 30%, 40%, 50%, 60% , 70%, 80%, 90% or 95% of the intrahepatic XO gene expression was suppressed in human subjects. In some embodiments, the siRNA concentration is 3 mg / kg, and the siRNA provided by the present disclosure The siRNA complex used in this study had a 7% suppression rate of XO mRNA expression in mice. 0.9-76.2%.

[0029] In some embodiments, the siRNA, drug composition or s The iRNA complexes did not show significant off-target effects. For example, the inhibition of normal expression of a non-target gene. Binding / inhibition was 50%, 40%, 30%, 20% or 1% lower than the on-target gene effect. 0% lower, the off-target effect is considered to be insignificant.

[0030] Thus, the siRNAs, drug compositions and siRNA complexes provided by the present disclosure and suppressing the expression of the XO gene, thereby preventing or treating uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders. It can be effectively treated and / or prevented, and has bright prospects for application.

[0031] Other features and advantages of the present disclosure are described in the Detailed Description section below. Let me explain in detail. [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] Hereinafter, the embodiments of the present disclosure will be described in detail. The detailed description is merely for the purpose of explaining or interpreting the present disclosure, and It should be understood that this is not intended to be limiting.

[0034] As used herein, XO mRNA is identified by Genbank accession number NM_000379 Further, unless otherwise indicated, in this disclosure, The term "target gene" as used herein refers to a gene that transcribes the XO mRNA. The term "target mRNA" refers to the XO mRNA.

[0035] <Definition> Unless otherwise stated in the context, the capital letters C, G, U, and A represent the base sequences of nucleotides. A lowercase letter m indicates that one nucleotide adjacent to the left of the letter m is modified with methoxy. A lowercase letter f indicates a nucleotide that is adjacent to the left of the letter f. The nucleotide is a fluoro-modified nucleotide, and the lowercase letter s is This indicates that two adjacent nucleotides are linked by a thiophosphate group. P1 is a nucleotide sequence in which one nucleotide adjacent to the right side of P1 is a 5'-phosphate nucleotide or represents a 5'-phosphate analog modified nucleotide, and the combination letter VP represents the The nucleotide immediately to the right of the combination VP is a vinyl phosphate-modified nucleotide. The combination letter Ps indicates that the combination letter Ps is a 1 adjacent letter to the right of the combination letter Ps. The capital letter P indicates that the nucleotide is a phosphorothioate modified nucleotide. The nucleotide immediately to the right of the letter P is a 5'-phosphate nucleotide. represent.

[0036] In this context, the term "fluoro-modified nucleotide" refers to a nucleotide having a ribose group of This refers to a nucleotide in which the 2'-hydroxy group is replaced with fluorine. "Leuotide" is a nucleotide in which the 2' hydroxyl group of the ribose group is replaced with a non-fluorine group. "Nucleotide analog" refers to a nucleotide or nucleotide analog that is Can substitute for nucleotides in nucleic acids, including adenine ribonucleotides, Guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or It refers to a group whose structure is different from thymine deoxyribonucleotide, for example, isonucleotide, Bridged nucleic acid (BNA) or acyclic nucleic acid The "methoxy-modified nucleotide" is a nucleotide that has a 2'-hydroxyl group at the ribose group. It refers to a nucleotide in which the oxy group is replaced with methoxy.

[0037] In the context of this specification, the terms "complement" and "reverse complement" are used interchangeably. It may have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, each of the bases of one strand is It means that the bases on one strand complement each other. The pyrimidine base adenine (A) always mates with the pyrimidine base thymine (T) (or In the nucleosome, uracil (U) pairs with the purine base guanine (C), which is always paired with the purine base guanine (C). It pairs with the lysine base cytosine (G). Each base pair consists of one purine and one It contains two pyrimidines: an adenine on one strand is always replaced by a thymine (or uracil) on the other strand. ) and guanine always pairs with cytosine, then both strands are complementary. It is also believed that the sequence of the strand can be deduced from the sequence of its complementary strand. In the art, the term "mismatch" refers to a situation in which bases at corresponding positions in a double-stranded nucleic acid are complementary. This means that there is no pairing of the two nuclei.

[0038] Unless otherwise specified in the context, "essentially reverse complementary" refers to the complementary structure of two related nucleotides. "Substantially reverse complementary" refers to the presence of three or fewer base mismatches between the nucleotide sequences. " refers to the presence of no more than one base mismatch between two nucleotide sequences; "Perfectly reverse complementary" means that there are no base mismatches between the two nucleotide sequences. Refers to...

[0039] In this context, one nucleotide sequence has a "nucleotide difference" with another nucleotide sequence. The presence of a "difference" means that the type of base at the same position in the former is different from that in the latter. For example, when one nucleotide base in the latter is A, the same If the corresponding nucleotide base at the same position is U, C, G, or T, Nucleotide differences between the two nucleotide sequences have been observed. In an embodiment, an abasic nucleotide or its equivalent is substituted for the nucleotide at the original position. When using the nucleotide sequence, it is considered that a nucleotide difference has occurred at that position.

[0040] In particular, in the context of the present disclosure, the method for preparing the siRNA, drug composition or siRNA complex In describing the above, unless otherwise specified, the nucleoside monomer e monomer) refers to the nucleoside in the siRNA or siRNA complex to be prepared. Depending on the type and order of phosphoramidites, modified or unmodified phosphoramidites are used in solid-phase phosphoramidite synthesis. Creoside phosphoramidite monomers (unmodified or modified d RNA phosphoramidites. RNA phosphoramid tes is sometimes called Nucleoside phosphoramidites) 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. do.

[0041] In the context of this specification, unless otherwise specified, "composite" means that each of the components has a specific function. refers to a covalent bond between two or more chemical moieties having A "complex" is a compound formed by covalently bonding each of the chemical moieties. Furthermore, an "siRNA complex" refers to a complex that contains one or more chemical moieties that have a specific function. In the following text, the term "siRNA" refers to a compound formed by covalently binding to an siRNA. The siRNA complex may also be referred to simply as a "complex." The siRNA complexes represented by the formulas (305) and (307) are generic names for the siRNA complexes. or the siRNA complexes represented by formula (305), (307), and (308). In the context of this specification, a "composite molecule" is understood to be a molecule that is formed by reacting and a specific molecule capable of being complexed with siRNA to ultimately form the siRNA complex of the present disclosure. It should be understood that the compound is a specific compound.

[0042] As used herein, "any" or "optionally" refers to the event or events described thereafter. that circumstances may or may not occur and that such statements are based on the assumption that an event or circumstance will occur. For example, "optionally substituted" "alkyl" refers to a group that may or may not occur. " includes "alkyl" and "substituted alkyl" as defined in the following sentence. With respect to any groups that contain substituents, it is important to note that these groups may be sterically impractical, synthetically impractical, or otherwise unfeasible. It is not intended to introduce any substitutions or substitution patterns that are unnecessary and / or inherently unstable. It will be understood by those skilled in the art that there is no.

[0043] As used herein, "alkyl" refers to both straight and branched chains having the specified number of carbon atoms. The specific number refers to a branched chain, and the specific number is usually 1 to 20 carbon atoms, for example, 1 to 10 carbon atoms. For example, C1-C6 alkyl is 1 to 6 Includes straight-chain and branched-chain alkyls of up to 100 carbon atoms. Refers to alkyl residues having a specific number of carbons. When used herein, it is intended to include all branched and straight chain forms having that number of carbons. Thus, for example, "butyl" includes n-butyl, sec-butyl, isobutyl and tert-butyl. -butyl, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl, just like alkyl, but with two points of attachment. It refers to a residue that

[0044] As used herein, "alkenyl" means an alkyl group having at least one carbon-carbon double bond. refers to an unsaturated branched or straight chain alkyl having a carbon-carbon double bond, The group is obtained by removing one hydrogen molecule from the adjacent carbon atom of the The double bonds may be in the cis or trans configuration. Exemplary alkenyls include vinyl and , prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1- propenyl, such as prop-2-en-2-yl, and but-1-en-1-yl. yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2- buta-1,3-dien-1-yl, buta-2-en-2-yl, buta-1,3-dien-1-yl, buta and butenyl, such as 1,3-dien-2-yl. In certain embodiments, alkenyl has 2 to 20 carbon atoms, while in other embodiments, Alkenylene has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. A subset of alkenyl, it refers to residues similar to alkenyl, but which have two points of attachment.

[0045] As used herein, "alkynyl" means an alkynyl group having at least one carbon-carbon triple bond. The term refers to an unsaturated branched or straight chain alkyl group having a carbon-carbon triple bond, It is obtained by removing two hydrogen molecules from adjacent carbon atoms of alkyl. The alkyl groups include ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, and prop-1-yn-1-yl. Propynyl, but-1-yn-1-yl, but-1-yn-3-yl, but- butynyl, such as 3-yn-1-yl. In certain embodiments, alkynyl has 2 to 20 carbon atoms, but in other embodiments, 2 to 1 Alkynylene is a subset of alkynyl having 0, 2 to 8, or 2 to 6 carbon atoms. and refers to a residue similar to alkynyl, but having two points of attachment.

[0046] As used herein, "alkoxy" refers to an alkyl group having a specified number of carbon atoms attached through oxygen bridges. alkyl of a carbon atom, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyl Oxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy Alkoxy is usually 1- having 10, 1-8, 1-6, or 1-4 carbon atoms bonded by oxygen bridges .

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

[0048] As used herein, a "halogen substituent" or "halo" refers to any group including 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 having a specified number of carbon atoms. is an alkyl group as defined above, substituted with up to the maximum permitted number of halogen atoms. Examples of halogenated alkyl groups include trifluoromethyl, difluoromethyl, and , 2-fluoroethyl, or pentafluoroethyl.

[0050] The term "heterocyclic group" refers to a group having 2 to 12 carbon atoms and 1 to 2 carbon atoms selected from nitrogen, oxygen, or sulfur. It refers to a stable 3- to 18-membered non-aromatic cyclic group containing 6 heteroatoms. Unless otherwise specified, heterocyclic groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, fused or bridged. The heteroatoms in the heterocyclic groups may be optionally oxidized. Nitrogen atoms, if present, are optionally quaternized. Heterocyclic groups may be partially saturated or completely Heterocyclic groups can be attached to the rest of the molecule through any atom of the ring. Examples of such heterocyclic groups include dioxanyl, thiophenyl[1,3]diphenyl. Sulfonyl (thienyl[1,3]dithianyl), decahydroisoquinolinyl Imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, Morpholinyl, Octahydroindole, Octahydroisoindole, 2-Oxapipe Razinyl, 2-oxapiperidyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl Dysyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidyl, quinuclidinyl , thiazolidinyl, tetrahydrofuryl, trisulfonyl, tetra thihydropyranyl, thiomorpholinyl, thiamol thiamorpholinyl, 1-oxothiomorpholinyl o-thiomorpholinyl) and 1,1-dioxothiomorpholinyl (1,1 -dioxo-thiomorpholinyl).

[0051] "Heteroaryl" means an alkyl group having 2 to 17 carbon atoms and a group selected from nitrogen, oxygen, and sulfur. refers to a group derived from a 3- to 18-membered aromatic ring radical containing 1 to 6 heteroatoms. As used herein, heteroaryl refers to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system. and at least one of the rings in the ring system is fully unsaturated, i.e., it is Heteroaryls include cyclic delocalized (4n+2) π-electron systems that conform to the Heteroaryl theory. includes bridged ring systems. The heteroatoms in heteroaryl are optionally oxidized. One or more The nitrogen atom (if present) is optionally quaternized. Heteroaryl is any of the heteroaryls in the ring. The atom that is attached to the rest of the molecule is azepinyl. acridinyl, benzimidazolyl, benzoindole, 1,3-benzodioxazoline aryl, benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl Benzo[b][1,4]dioxepinyl pinyl), benzo[b][1,4]oxazinyl azinyl), 1,4-benzodioxanyl, Benzonaphthofuranyl, benzoxazolyl, benzodioxolyl olyl), 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-dihydro benzo[h]quinazolinyl), 5,6-dihydrobenzo[h]quinazolinyl 5,6 dihydrobenzo[h]cinnolinyl, 6,7-dihydrobenzo[h]cinnolinyl Doro-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 Indazolyl, indole, isoindole, indolinyl, isoindolinyl nyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-Methano-5,6,7,8-tetrahydroquinazolinyl -5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl (naphthyridinyl), 1,6-naphthyridinonyl (1,6-naphth yridinonyl, oxadiazolyl, 2-oxoazepinyl pinyl), oxazolyl, oxiranyl, 5,6,6a,7, 8,9,10,10a-Octahydrobenzo[H]quinazolinyl, 1-phenyl-1H- Pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl (pht halazinyl, pteridinyl, purinyl, pyrrolyl, Pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridyl, pyrido[3,2-d]pyridinyl Rimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl nyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl 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, thiadia Zolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl Thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl o[2,3-c]pridinyl) and thiophenyl / thi enyl), but are not limited to

[0052] A variety of hydroxy protecting groups may be used in the present disclosure. Generally, the protecting groups are In this way, chemical functionality can be rendered insensitive to certain reaction conditions while leaving the remainder of the molecule substantially can be added to and removed from that functionality in a molecule without physical damage A representative hydroxy protecting group is described in Beaucage et al., Tetrahedron 19 92, 48, 2223-2311, and Greene and Wuts, Protect tive Groups in Organic Synthesis,Chapter 2,2d ed., John Wiley & Sons, New York, 1991 No. 6,393,962, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions but is stable under acidic conditions. In some embodiments, the hydroxy groups usable herein can be removed. Non-exclusive examples of protecting groups include dimethoxytrityl (DMT), monomethoxytrityl , 9-phenylxanthen-9-yl (Pixyl) or 9-(p-methoxyphenyl) In some embodiments, the compounds used herein include xanthen-9-yl (Mox). Non-exclusive examples of hydroxy protecting groups that can be used include Tr (trityl), MMTr (4-methyl-2-phenylpropanediol), 4,4'-dimethoxytrityl), 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. Subjects of the present disclosure include humans, non-human primates (e.g., monkeys or other species of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats or any type of poultry, but is not limited to such.

[0054] As used herein, "treatment" refers to a method for obtaining beneficial or desired results; "Therapeutic effect" includes, but is not limited to, the eradication or elimination of the underlying disorder being treated. Treatment efficacy means that the subject is still suffering from the underlying disorder. Although the patient may be at risk of suffering from one or more physiological symptoms associated with the underlying disorder, By eliminating or improving, an improvement is observed in the subject.

[0055] As used herein, "prophylaxis" refers to a method of obtaining a beneficial or desired result, and includes prevention. In order to obtain a "preventive effect," the diagnosis of the disease in question is made. Although no cutting may have been performed, the siRNA, siRNA complex, or drug composition may be identified. The present invention relates to a method for treating a subject at risk of developing a disease, or for treating one or more pathological symptoms of the disease. The subject may be administered the drug.

[0056] In one aspect, the present disclosure provides first to twelfth siRNAs capable of suppressing XO gene expression. Each of these will be explained in detail below.

[0057] The siRNA of the present disclosure comprises a nucleotide group as a basic structural unit. It is known to those skilled in the art that a phosphate group includes a ribose group and a base. The explanation will be omitted.

[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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 form a region, The nucleotide sequence is the same in length and has no more than three nucleotide differences, and is identical to the nucleotide sequence II of SEQ ID NO: The nucleotide sequence shown in No. 2 is the same length and has no more than three nucleotide differences. do. 5'-GAGAUGAAGUUCAAGAAUZ1-3' (SEQ ID NO: 1), 5'-Z2AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 2) wherein Z1 is A and Z2 is U, and in said nucleotide sequence I, position Z1 is The corresponding nucleotide Z3 is included in the nucleotide sequence II, and the position of Z2 is included in the nucleotide sequence II. and Z4 is the first nucleotide at the 5' end of the antisense strand. It is a nucleotide.

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

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

[0062] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 and / or the nucleotide sequence has no more than one nucleotide difference There is not more than one nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:2. be.

[0063] In some embodiments, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 Nucleotide differences between the nucleotide sequences include differences at position Z4, where Z4 is A, C or is selected from G. In some embodiments, the nucleotide difference is and Z4 is selected from A, C, or G. Z3 is a nucleotide complementary to Z4. siRNAs having the above nucleotide differences The siRNAs containing these nucleotide differences also have high target mRNA silencing ability. within the scope of protection of the present disclosure.

[0064] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , essentially reverse complementary, substantially reverse complementary or completely reverse complementary, "targeted" refers to the presence of three or fewer base mismatches between two nucleotide sequences; The term "substantially reverse complementary" means that there is not more than one base mismatch between the two nucleotide sequences. Perfectly reverse-complementary means that there are no base mismatches between the two nucleotide sequences. It refers to something that does not exist.

[0065] In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:3. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:4. do. 5'-GAGAUGAAGUUCAAGAAUZ3-3' (SEQ ID NO: 3), 5'-Z4AUUCUUGAACUUCAUCUC-3' (SEQ ID NO: 4) However, Z4 is the first nucleotide at the 5' end of the antisense strand, and Z4 is selected from A, U, G or C, Z3 is a nucleotide complementary to Z4, In some embodiments, Z3 is A and Z4 is U.

[0066] The sense strand and the antisense strand may have the same or different lengths, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure 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, 2 0 / 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, 2 In some embodiments, the front The length ratio of the sense strand to the antisense strand of the siRNA was 19 / 21, 21 / 23, or 23 / It is 25.

[0067] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence. is adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO:1 in the target mRNA, It refers to a nucleotide sequence equal in length to nucleotide sequence IV above.

[0068] In some embodiments, nucleotide sequence III and nucleotide sequence IV , each of which is 1 nucleotide in length, the base of nucleotide sequence III is U, The base of nucleotide sequence IV is A, and the length ratio of the sense strand to the antisense strand is 20 / 20, or nucleotide sequences III and IV are both 2 nucleotides in length. The nucleotide sequence of nucleotide sequence III from the 5' end to the 3' end is UU, and the base sequence of nucleotide sequence IV is AA, in which case the sense strand and the antisense strand are The length ratio of the sense strand is 21 / 21, or the nucleotide sequences III and IV are The length of each is 3 nucleotides, and the nucleotide sequence is the base sequence of nucleotide sequence III is AUU and the base sequence of nucleotide sequence IV is AAU; In this case, the length ratio of the sense strand to the antisense strand is 22 / 22, or Sequences III and IV are both 4 nucleotides in length and extend 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 The base sequence is AAUG, and the length ratio of the sense strand to the antisense strand is 23 / 2 In some embodiments, the nucleotide sequence III is Column IV is 2 nucleotides in length and is the base sequence of nucleotide sequence III is UU and the base sequence of nucleotide sequence IV is AA; In this case, 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 Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61 form a region. , which are equal in length and have no more than three nucleotide differences, and which are identical to nucleotide sequence II and sequence The nucleotide sequence shown in FIG. 62 is the same length as the nucleotide sequence shown in FIG. 62, and differs by no more than three nucleotides. It is. 5'-CAUAACUGGAAUUUGUAAZ5-3' (SEQ ID NO:61), 5'-Z6UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:62) wherein Z5 is U and Z6 is A; and in said nucleotide sequence I, position Z5 is The corresponding nucleotide Z7 is included in the nucleotide sequence II, which corresponds to Z6 in position II. the antisense strand includes a nucleotide Z8 which is the first nucleotide at the 5' end of the antisense strand; It is a nucleotide.

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

[0073] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61 and / or There is one or more nucleotide differences between Sequence II and the nucleotide sequence shown in SEQ ID NO:62. Below.

[0074] In some embodiments, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:62 Nucleotide differences between the nucleotide sequences include a difference at position Z8, where Z8 is U, C or G. In some embodiments, the nucleotide difference is selected from Z In some embodiments, the difference is in position Z8, and Z is selected from U, C, or G. Z7 is the nucleotide complementary to Z8. A has high target mRNA suppression ability, and siRNAs containing these nucleotide differences , are within the scope of protection of this disclosure.

[0075] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. Nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:64. It is. 5'-CAUAACUGGAAUUUGUAAZ7-3' (SEQ ID NO: 63), 5'-Z8UUACAAAUUCCAGUUAUG-3' (SEQ ID NO:64) However, Z8 is the first nucleotide at the 5' end of the antisense strand, and Z8 is selected from A, U, G or C; Z7 is a nucleotide complementary to Z8; In some embodiments, Z7 is U and Z8 is A.

[0077] The sense strand and the antisense strand may have the same or different lengths, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0078] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. The nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely complementary to each other. The second nucleotide sequence is a sequence number 1 in the target mRNA. 61, the length of which is adjacent to the 5' end of the nucleotide sequence shown in V.

[0079] In some embodiments, nucleotide sequence III and nucleotide sequence IV , each of which is one nucleotide in length, the base of nucleotide sequence III is A, The base of nucleotide sequence IV is U, and the length ratio of the sense strand to the antisense strand is 20 / 20, or nucleotide sequences III and IV are both 2 nucleotides in length. The nucleotide sequence of nucleotide sequence III from the 5' end to the 3' end is AA, and the base sequence of nucleotide sequence IV is UU. In this case, the sense strand and the antisense strand are The length ratio of the sense strand is 21 / 21, or the nucleotide sequences III and IV are The length of each is 3 nucleotides, and the nucleotide sequence is the base sequence of nucleotide sequence III is UAA and the base sequence of nucleotide sequence IV is UUA; In this case, the length ratio of the sense strand to the antisense strand is 22 / 22, or Sequences III and IV are both 4 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is GUAA, and the base sequence of nucleotide sequence IV is The base sequence is UUAC, and the length ratio of the sense strand to the antisense strand is 23 / 2 In some embodiments, the nucleotide sequence III is Column IV is 2 nucleotides in length and is the base sequence of nucleotide sequence III is AA and the base sequence of nucleotide sequence IV is UU; In this case, 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 Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 form a region. are equal in length, have no more than three nucleotide differences, and are identical to nucleotide sequence II. The nucleotide sequence shown in column 122 is the same length and has three nucleotide differences. The following is the result. 5'-CAUUAUCACAAUUGAGGAZ9-3' (SEQ ID NO: 121), 5'-Z 10 UCCUCAAUUGUGAUAAUG-3' (SEQ ID NO: 122) However, Z9 is U and Z 10 is A, and in said nucleotide sequence I, position Z9 Nucleotide Z corresponding to 11 and in said nucleotide sequence II, position Z 10 To The corresponding nucleotide Z 12 wherein Z 12 is the 5' end of the antisense strand This is the first nucleotide.

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

[0084] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:121 and / or There is one nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:122. There are less than one.

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

[0086] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:124. It is an array. 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 is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0089] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. The nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely complementary to each other. The second nucleotide sequence is a sequence number 1 in the target mRNA. 121, the length of which is adjacent to the 5' end of the nucleotide sequence shown in It refers to the nucleotide sequence equivalent to IV.

[0090] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is C and the base of nucleotide sequence IV is G, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged from the 5' end to the 3' end. the base sequence of nucleotide sequence III is GC, the base sequence of nucleotide sequence IV is GC, In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is AGC, and the base sequence of nucleotide sequence IV is The base sequence 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. The base sequence of nucleotide sequence IV is GCUG, and the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is GC, and the base sequence of nucleotide sequence IV is The base sequence is GC, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0091] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 form a region. are equal in length, have no more than three nucleotide differences, and are identical to nucleotide sequence II. The nucleotide sequence shown in column 182 is the same length and has three nucleotide differences. The following is the result. 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 Z1 Nucleotide Z corresponding to 4 16 wherein Z 16 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0095] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:181 and / or There is a 1 nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:182. There are less than one.

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

[0097] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:184. It is an array. 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 is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0100] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. The nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely complementary to each other. The second nucleotide sequence is a sequence number 1 in the target mRNA. 181, the length of which is adjacent to the 5' end of said nucleotide sequence It refers to the nucleotide sequence equivalent to IV.

[0101] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is A and the base of nucleotide sequence IV is U, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged 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; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is CCA, and the base sequence of nucleotide sequence IV is The base sequence 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. The base sequence of nucleotide sequence IV is UGGG, and the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is CA, and the base sequence of nucleotide sequence IV The base sequence is UG, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0102] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:241 form a region. are equal in length, have no more than three nucleotide differences, and are identical to nucleotide sequence II. The nucleotide sequence shown in column 242 is the same length and has three nucleotide differences. The following is the result. 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 Z1 Nucleotide Z corresponding to 8 20 wherein Z 20 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0106] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:241 and / or There is 1 nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:242. There are less than one.

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

[0108] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:244. It is an array. 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 is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0111] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. The nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely complementary to each other. The second nucleotide sequence is a sequence number 1 in the target mRNA. 241, the length of which is adjacent to the 5' end of the nucleotide sequence shown in It refers to the nucleotide sequence equivalent to IV.

[0112] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is C and the base of nucleotide sequence IV is G, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged 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; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is UCC, and the base sequence of nucleotide sequence IV is The base sequence 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; The base sequence of nucleotide sequence III from the 5' end to the 3' end is UUCC. The base sequence of nucleotide sequence IV is GGAA. In this case, the sense strand and the antisense strand In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is CC, and the base sequence of nucleotide sequence IV is The base sequence is GG, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0113] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 form a region. are equal in length, have no more than three nucleotide differences, and are identical to nucleotide sequence II. The nucleotide sequence shown in column 302 is the same length and has three nucleotide differences. The following is the result. 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 Z2 Nucleotide Z corresponding to 2 24 wherein Z 24 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0117] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 are and / or There is one nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:302. There are less than one.

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

[0119] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:304. It is an array. 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 is a nucleotide complementary to In some embodiments, Z 23is A and Z 24 is U.

[0121] The sense strand and the antisense strand may have the same or different lengths, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0122] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. The nucleotide sequence IV and the second nucleotide sequence are substantially reverse complementary or completely complementary to each other. The second nucleotide sequence is a sequence number 1 in the target mRNA. 301, the length of which is adjacent to the 5' end of the nucleotide sequence shown in It refers to the nucleotide sequence equivalent to IV.

[0123] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is C and the base of nucleotide sequence IV is G, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged 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; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is GCC, and the base sequence of nucleotide sequence IV is The base sequence 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. The base sequence of nucleotide sequence IV is GGCA. In this case, the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is CC, and the base sequence of nucleotide sequence IV is The base sequence is GG, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0124] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 form a region. are equal in length, have no more than three nucleotide differences, and are identical to nucleotide sequence II. The nucleotide sequence shown in column 362 is the same length and has three nucleotide differences. The following is the result. 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 Z2 Nucleotide Z corresponding to 6 28 wherein Z 28 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0128] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:361 and / or There is 1 nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:362. There are less than one.

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

[0130] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:364. It is an array. 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 28is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0133] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence. adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO: 361 in the target mRNA and refers to a nucleotide sequence having the same length as nucleotide sequence IV.

[0134] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is G and the base of nucleotide sequence IV is C, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged from the 5' end to the 3' end. the base sequence of nucleotide sequence III is GG and the base sequence of nucleotide sequence IV is CC; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is AGG, and the base sequence of nucleotide sequence IV is The base sequence 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; From the 5' end to the 3' end, the base sequence of nucleotide sequence III is AAGG. The base sequence of nucleotide sequence IV is CCUU, and the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is GG, and the base sequence of nucleotide sequence IV is The base sequence is CC, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0135] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421 form a region. are equal in length, have no more than three nucleotide differences, and are identical to nucleotide sequence II. The nucleotide sequence shown in column 422 is the same length and has three nucleotide differences. The following is the result. 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 Z3 Nucleotide Z corresponds to 0 32 wherein Z 32 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0139] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:421 and / or There is one nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:422. There are less than one.

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

[0141] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. The nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 424. It is a chid sequence. 5'-GAAAAUCACCUAUGAAGAZ 31 -3' (SEQ ID NO: 423), 5'-Z 32 UCUUCAUAGGUGAUUUUC-3' (SEQ ID NO: 424) However, the above Z 32is 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 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0144] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence. adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO: 421 in the target mRNA and refers to a nucleotide sequence having the same length as nucleotide sequence IV.

[0145] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is U and the base of nucleotide sequence IV is A, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged from the 5' end to the 3' end. the base sequence of nucleotide sequence III is GU, the base sequence of nucleotide sequence IV is AC, In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is GGU, and the base sequence of nucleotide sequence IV is GGU. The base sequence 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. The base sequence of nucleotide sequence IV is ACCC, and the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is GU, and the base sequence of nucleotide sequence IV is The base sequence is AC, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0146] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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, wherein each nucleotide is independently a modified or unmodified nucleotide, and the sense strand comprises the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are double-stranded in a reverse complementary manner at least in part. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 481 form a region. are equal in length, have no more than three nucleotide differences, and are identical to nucleotide sequence II. The nucleotide sequence shown in column 482 is the same length and has three nucleotide differences. The following is the result. 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 Z3 Nucleotide Z corresponding to 4 36 wherein Z 36 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0150] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:481 and / or There is 1 nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:482. There are less than one.

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

[0152] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:484. It is an array. 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 is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0155] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence. adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO: 481 in the target mRNA and refers to a nucleotide sequence having the same length as nucleotide sequence IV.

[0156] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is G and the base of nucleotide sequence IV is C, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged from the 5' end to the 3' end. the base sequence of nucleotide sequence III is AG and the base sequence of nucleotide sequence IV is CU; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend 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 The base sequence 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' end to the 3' end, the base sequence of nucleotide sequence III is UGAG. The base sequence of nucleotide sequence IV is CUCA, and the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is AG, and the base sequence of nucleotide sequence IV is The base sequence is CU, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0157] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are 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 said sense amino acid sequence is independently a modified or unmodified nucleotide; the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are reverse-complementary to each other at least in part. A double-stranded region is formed, and the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:541 are The sequences are of equal length and have no more than three nucleotide differences, and are and the nucleotide sequence shown in SEQ ID NO:542 are equal in length and have a nucleotide difference of Three or less. 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 Z3 Nucleotide Z corresponding to 8 40 wherein Z 40 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0161] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:541 and / or There is 1 nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:542. There are less than one.

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

[0163] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:544. It is an array. 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 is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0166] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence. adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO:541 in the target mRNA and refers to a nucleotide sequence having the same length as nucleotide sequence IV.

[0167] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is A and the base of nucleotide sequence IV is U, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged 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; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is AAA, and the base sequence of nucleotide sequence IV is The base sequence 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. The base sequence of nucleotide sequence IV is UUUA. In this case, the sense strand and the antisense strand In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is AA, and the base sequence of nucleotide sequence IV is The base sequence is UU, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0168] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are also determined.

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

[0170] The eleventh siRNA comprises a sense strand and an antisense strand, each nucleotide of said sense amino acid sequence is independently a modified or unmodified nucleotide; the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are reverse-complementary to each other at least in part. A double-stranded region is formed, and the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:601 are The sequences are of equal length and have no more than three nucleotide differences, and are and the nucleotide sequence shown in SEQ ID NO:602 are equal in length and have a nucleotide difference of Three or less. 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 Z4 Nucleotide Z corresponding to 2 44 wherein Z 44 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0172] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:601 are and / or There is one nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:602. There are less than one.

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

[0174] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:604. It is an array. 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 is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0177] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence. adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO: 601 in the target mRNA and refers to a nucleotide sequence having the same length as nucleotide sequence IV.

[0178] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is G and the base of nucleotide sequence IV is C, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged from the 5' end to the 3' end. the base sequence of nucleotide sequence III is GG and the base sequence of nucleotide sequence IV is CC; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend 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 The base sequence 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; From the 5' end to the 3' end, the base sequence of nucleotide sequence III is GUGG. The base sequence of nucleotide sequence IV is CCAC, and the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is GG, and the base sequence of nucleotide sequence IV is The base sequence is CC, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0179] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are also determined.

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

[0181] The twelfth siRNA comprises a sense strand and an antisense strand, each nucleotide of said sense amino acid sequence is independently a modified or unmodified nucleotide; the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, The nucleotide sequence I and the nucleotide sequence II are reverse-complementary to each other at least in part. A double-stranded region is formed, and the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:661 are The sequences are of equal length and have no more than three nucleotide differences, and are and the nucleotide sequence shown in SEQ ID NO:662 are equal in length and have a nucleotide difference of Three or less. 5'-CUUCUUUGCCAUCAAAGAZ 45 -3' (SEQ ID NO: 661), 5'-Z 46 UCUUUGAUGGCAAAGAAG-3' (SEQ ID NO:662) However, Z 45 is U and Z46 is A, and in said nucleotide sequence I, position Z 45 Nucleotide Z corresponding to 47 and in said nucleotide sequence II, position Z4 Nucleotide Z corresponding to 6 48 wherein Z 48 is the 5' end of the antisense strand It is the first nucleotide at the end.

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

[0183] In some embodiments, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:661 and / or There is 1 nucleotide difference between sequence II and the nucleotide sequence shown in SEQ ID NO:662. There are less than one.

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

[0185] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , 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. nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:664. It is an array. 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 is a nucleotide complementary to 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, and the length of the sense strand is The length of the antisense strand is 19-26 nucleotides. .

[0188] In some embodiments, the sense strand further comprises nucleotide sequence III. the antisense strand further comprises nucleotide sequence IV; and 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 substantially reverse-complementary or completely complementary. and said nucleotide sequence III is a reverse complement to said nucleotide sequence I at the 5' end thereof. and said nucleotide sequence IV is linked to the 3' end of said nucleotide sequence II. In some embodiments, the nucleotide sequence IV and the second nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence. adjacent to the 5' end of the nucleotide sequence set forth in SEQ ID NO: 661 in the target mRNA and refers to a nucleotide sequence having the same length as nucleotide sequence IV.

[0189] In some embodiments, the nucleotide sequence III is and nucleotide sequence IV are each one nucleotide in length, and nucleotide sequence I The base of II is U and the base of nucleotide sequence IV is A, then the sense strand The length ratio of the antisense strand is 20 / 20, or the nucleotide sequences III and IV are Each has a length of 2 nucleotides, and the nucleotides are arranged from the 5' end to the 3' end. the base sequence of nucleotide sequence III is AU, the base sequence of nucleotide sequence IV is AU, In this case, the length ratio of the sense strand to the antisense strand is 21 / 21, or Sequences III and IV are both 3 nucleotides in length and extend 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 UAU. The base sequence 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' end to the 3' end, the base sequence of nucleotide sequence III is CUAU. The base sequence of nucleotide sequence IV is AUAG, and the sense strand and the antisense strand are In some embodiments, the nucleotide sequence is 23 / 23. String III and nucleotide sequence IV are 2 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is AU, and the base sequence of nucleotide sequence IV is The base sequence is AU, and the length ratio of the sense strand to the antisense strand is 21 / 21. be.

[0190] In some embodiments, nucleotide sequence III and nucleotide sequence IV are Since it is fully reverse complementary, given the base of nucleotide sequence III, The bases of sequence IV are also determined.

[0191] The following describes the nucleotide sequence V, the nucleic acid sequence, the nucleotide modifications in the siRNA, and The explanation of the modified sequence is applicable to any one of the above 1st to 12th siRNAs. That is, unless otherwise specified, the following description of siRNAs refers to the first siRNA, the second siRNA, 1 siRNA, 3 siRNA, 4 siRNA, 5 siRNA, 6 siRNA NA, 7th siRNA, 8th siRNA, 9th siRNA, 10th siRNA, The 11th and 12th siRNAs should be considered as separate descriptions. For example, if a particular siRNA is not specified, "the siRNA is a nucleic acid "further comprising an octide sequence V" means "a first siRNA, a second siRNA, a third siRNA, RNA, a fourth siRNA, a fifth siRNA, a sixth siRNA, a seventh siRNA, The eighth siRNA, the ninth siRNA, the tenth siRNA, the eleventh siRNA or the 12 siRNAs further comprise nucleotide sequence V."

[0192] In some embodiments, the antisense strand further comprises the nucleotide sequence V. nucleotide sequence V is 1 to 3 nucleotides in length, and the 3 The 3' end of the antisense strand is bound to the 3' end, forming the 3' overhanging end of the antisense strand. As a result, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure is 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 / 2 In some embodiments, the nucleotide sequence V is 2 nucleotides in length, thereby defining the siRNA of the present disclosure. The ratio of sense to antisense strand lengths is 19 / 21, 21 / 23, or 23 / 25. good.

[0193] Each nucleotide in the nucleotide sequence V may be any nucleotide. In order to reduce the cost of synthesis, the nucleotide sequence V is preferably composed of two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides Alternatively, the affinity of the antisense strand of the siRNA to the target mRNA is To improve potency, the nucleotide sequence V is a nucleotide sequence corresponding to the nucleotide sequence of the target mRNA. Thus, in some embodiments, the siRNA of the present disclosure The ratio of the length of the sense strand to the antisense strand is 19 / 21 or 21 / 23. The disclosed siRNAs have superior target mRNA silencing activity.

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

[0195] In some embodiments, the siRNA is The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:5. The strand comprises the nucleotide sequence shown in SEQ ID NO:6. 5'-GAGAUGAAGUUCAAGAAUZ3-3' (SEQ ID NO:5), 5'-Z4AUUCUUGAACUUCAUCUCAA-3' (SEQ ID NO: 6)

[0196] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:7. The antisense strand comprises the nucleotide sequence shown in SEQ ID NO:8. 5'-UUGAGAUGAAGUUCAAGAAUZ3-3' (SEQ ID NO: 7), 5'-Z4AUUCUUGAACUUCAUCUCAAUG-3' (SEQ ID NO: 8) However, Z4 is the first nucleotide at the 5' end of the antisense strand, and Z4 is selected from A, U, G or C, and Z3 is the nucleotide complementary to Z4.

[0197] In some embodiments, the siRNA is fused to the second siRNA. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:65. The base strand comprises the nucleotide sequence shown in SEQ ID NO:66. 5'-CAUAACUGGAAUUUGUAAZ7-3' (SEQ ID NO: 65), 5'-Z8UUACAAAUUCCAGUUAUGUU-3' (SEQ ID NO:66)

[0198] Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:67. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:68. nothing. 5'-AACAUAACUGGAAUUUGUAAZ7-3' (SEQ ID NO: 67), 5'-Z8UUACAAAUUCCAGUUAUGUUAC-3' (SEQ ID NO:68) However, Z8 is the first nucleotide at the 5' end of the antisense strand, and Z8 is selected from A, U, G or C, and Z7 is the nucleotide complementary to Z8.

[0199] In some embodiments, the siRNA is a nucleotide sequence of the siRNA relative to the third siRNA. The siRNA strand comprises the nucleotide sequence shown in SEQ ID NO: 125. The sense strand comprises the nucleotide sequence shown 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. wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:128. Includes. 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 do.

[0201] In some embodiments, the siRNA is The siRNA strand comprises the nucleotide sequence shown in SEQ ID NO: 185. The sense strand 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. wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:188. Includes. 5'-CAGGAUCUCUCUCAGAGUAUZ 15 -3' (SEQ ID NO: 187), 5'-Z 16 AUACUCUGAGAGAGAUCCUGGG-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 Z16 is a nucleotide complementary to do.

[0203] In some embodiments, the siRNA is The siRNA strand comprises the nucleotide sequence shown in SEQ ID NO: 245. The sense strand 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. wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 248. Includes. 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 do.

[0205] In some embodiments, the siRNA is The nucleotide strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 305. The sense strand comprises the nucleotide sequence set forth in SEQ ID NO:306. 5'-UAGCAAGCUCUCAGUAUCZ23 -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. wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 308. Includes. 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 do.

[0207] In some embodiments, the seventh siRNA is The nucleotide strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 365. The sense strand 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 shown in SEQ ID NO: 367. wherein the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:368. 5'-GGAUAAGGUUACUUGUGUUGZ27 -3' (SEQ ID NO: 367), 5'-Z 28 CAACACAAGUAACCUUAUCCUU-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 do.

[0209] In some embodiments, the siRNA is The siRNA strand comprises the nucleotide sequence shown in SEQ ID NO: 425. The sense strand 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 shown in SEQ ID NO: 427. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:428. Includes. 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 do.

[0211] In some embodiments, the ninth siRNA is The nucleotide strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 485. The sense strand 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. wherein the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:488. Includes. 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 do.

[0213] In some embodiments, the sequence of the 10th siRNA is The antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 545. The sense strand 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. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:548. Includes. 5'-AAGAACAACUCCUUUUAUGGZ 39 -3' (SEQ ID NO: 547), 5'-Z 40 CCAUAAAAGGAGUUGUUCUUUA-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 do.

[0215] In some embodiments, the 11th siRNA is The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 605. The sense strand 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. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:608. Includes. 5'-GGCUUGCUCUGAAGUAGAAAZ43 -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 do.

[0217] In some embodiments, the 12th siRNA is selected from the group consisting of siRNAs. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 665. The sense strand 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. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:668. Includes. 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 do.

[0219] In some embodiments, the siRNA described in this disclosure is selected from the group consisting of siRNAs described in Tables 1a-1l. siXOa1, siXOa2, siXOb1, siXOb2, and siXOc1 are listed , siXOc2, siXOd1, siXOd2, siXOe1, siXOe2, siXO f1, siXOf2, siXOg1, siXOg2, siXOh1, siXOh2, si XOi1, siXOi2, siXOj1, siXOj2, siXOk1, siXOk2, Either siXOl1 or siXOl2.

[0220] As discussed above, each nucleotide in the siRNA of the present disclosure may be independently modified or is an unmodified nucleotide. In some embodiments, each of the siRNAs of the present disclosure All nucleotides are unmodified nucleotides. Some or all of the nucleotides in the disclosed siRNAs are modified nucleotides. These modifications on the nucleotide group allow the disclosed siRNA complexes to suppress XO gene expression. There is no appreciable weakening or loss of the inhibitory function.

[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 A nucleotide or nucleotide in which the 2' hydroxyl group of the ribose group of the nucleotide has been replaced with another group. The term "modified nucleotide" refers to a nucleotide analog or a nucleotide having a modified base. The nucleotides clearly weakened or abolished the function of siRNA in suppressing gene expression. For example, JK Watts, GF Deleavey, and MJ Damha,Chemically modified siRNA: t ools and applications. Drug Discov Today ,2008,13(19-20): 842-55 You may do so.

[0222] In some embodiments, the sense strand or the siRNA provided by the present disclosure The antisense strand has at least one modified nucleotide, and / or At least one of the phosphate ester groups is a phosphate ester group having a modifying group. In other words, at least one single stranded phosphate in the sense strand and the antisense strand At least some of the phosphate groups and / or ribose groups in the sugar backbone have a modified group. The ribose group may be a phosphate group and / or a modified group.

[0223] In some embodiments, the nucleic acid in the sense strand and / or the antisense strand In some embodiments, the nucleotides are all modified nucleotides. Each nucleotide in the sense strand and the antisense strand of the siRNA provided herein is unique. They are either fluoro-modified or non-fluoro-modified nucleotides.

[0224] The inventors of the present disclosure have surprisingly found that the siRNAs described in the present disclosure can be used in animal studies. Achieve a high balance between plasma stability and gene silencing efficiency in found.

[0225] In some embodiments, the fluoro-modified nucleotide is and located in nucleotide sequence II, from the 5' end to the 3' end, At least the nucleotides at positions 7, 8, and 9 of sequence I are fluoro-modified nucleotides; From the 5' end to the 3' end, at least the second, sixth, first and The nucleotides at positions 4 and 16 are fluoro-modified nucleotides.

[0226] In some embodiments, the fluoro-modified nucleotide is and a fluoro-modified nucleotide in nucleotide sequence I located at nucleotide sequence II; nucleotide sequence I, At least the nucleotides at positions 7, 8, and 9 are fluoro-modified nucleotides, The fluoro-modified nucleotides in nucleotide sequence II are 7 or less, At least the 2nd, 6th, 14th and 16th nucleotides of sequence II are fluoro-modified nucleotides. It's Chid.

[0227] In some embodiments, from the 5' end to the 3' end, in the sense strand, and the nucleotides at positions 7, 8, and 9 or 5, 7, 8, and 9 of the nucleotide sequence I are and the remaining nucleotides in the sense strand are non-fluoro-modified nucleotides. the antisense strand is a fluoro-modified nucleotide from the 5' end to the 3' end In the nucleotide sequence II, The nucleotides at positions 4 and 16 are fluoro-modified nucleotides in the antisense strand. The nucleotides at the remaining positions are non-fluoro modified nucleotides.

[0228] In the context of this specification, a "fluoro-modified nucleotide" refers to a ribosomal nucleotide of a nucleotide. The hydroxy group at the 2' position of the aryl group is substituted with fluorine. "Non-fluoro modified nucleotide" refers to a nucleotide that is A nucleotide in which the 2'-hydroxy group of the nucleotide group is replaced with a non-fluorine group, or In some embodiments, each non-fluoro modified nucleotide is a nucleotide analog. A nucleotide or nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced with a non-fluorine group. is one independently selected from the nucleotide analogs.

[0229] Nucleotides in which the 2' hydroxyl group of the ribose group is replaced with a non-fluorine group are , known to those skilled in the art, and these nucleotides include 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 The nucleic acid may be one selected from the group consisting of nucleotides, nucleotides, and 2'-deoxynucleotides.

[0230] In some embodiments, the 2'-alkoxy modified nucleotide is shown in formula (8): In some embodiments, the 2'-OMe nucleotide is a methoxy modified nucleotide (2'-OMe). The 2'-substituted alkoxy modified nucleotide is, for example, a 2'-O-methionyl nucleotide represented by formula (9): In some embodiments, the nucleotides may be 2'-oxyethyl modified nucleotides (2'-MOE). In the present invention, the 2'-amino modified nucleotide (2'-NH2) is shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is represented by formula (11): .

[0231] [ka]

[0232] Nucleotide analogs are compounds that can replace nucleotides in nucleic acids. , adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, Refers to a group whose structure is different from that of uracil ribonucleotide or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog is an isonucleotide, a bridged nucleotide, Bridged nucleic acid (BNA) or acyclic nucleic acid It may be a do.

[0233] BNA refers to constrained or inaccessible nucleotides. BNAs are five-membered, six-membered The cyclic or seven-membered rings are bridged with a "fixed" C3'-endo sugar puckering. Typically, the bridge is introduced at the 2'- and 4'-positions of the ribose to form 2',4'-BN. In some embodiments, the BNA is provided as shown in formula (12): LNA shown in formula (13), ENA shown in formula (14), cET BNA shown in formula (14), etc. This is also possible.

[0234] [ka]

[0235] Acyclic nucleotides are nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide is an unlocked nucleic acid represented by formula (15): (UNA), or glycerol nucleic acid (GNA) as shown in formula (16).

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

[0237] Isonucleotides are nucleotides in which the position of the base in the ribose ring changes. In some embodiments, the isonucleotide is represented by the formula (17) or ( 18), in which the base is transferred from the 1'-position to the 2'- or 3'-position of the ribose ring. It may also be a mixture.

[0238] [ka] In the compounds of the above formulas (17) and (18), Base is A, U, G, C, T, etc. wherein R is selected from H, OH, F or a non-fluorine group as described above.

[0239] In some embodiments, the nucleotide analog is an isonucleotide, an LNA, In some embodiments, the IL-16 receptor is one selected from ENA, cET, UNA, and GNA. In the present study, each non-fluoro-modified nucleotide was a methoxy-modified nucleotide. In the context, the methoxy-modified nucleotide is a nucleotide in which the 2'-hydroxy group of the ribose group is methylated. It refers to a nucleotide substituted with toxin.

[0240] In the context, "fluoro-modified nucleotide", "2'-fluoro-modified nucleotide" "Nucleotides in which the 2'-hydroxy group of the ribose group is replaced with fluorine" and "2'- "Nucleotide having a fluororibose group" and "Nucleotide having a fluororibose group" have the same meaning. A compound having the structure shown in formula (7) in which the 2'-hydroxy group of Refers to "methoxy-modified nucleotide," "2'-methoxy-modified nucleotide," and "ribo "Nucleotides in which the 2'-hydroxy group of the 2'-hydroxyl group is substituted with methoxy" and "2'-Methoxy "Nucleotide having a silibose group" and "nucleotide having a silibose group" have the same meaning. The 2'-hydroxy group of the bosyl group is replaced with methoxy, and the structure is shown in formula (8). This refers to a compound that

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

[0242] In some embodiments, the siRNA of the present disclosure has the following modifications: That is, from the 5' end to the 3' end, The nucleotides at positions 5, 7, 8 and 9 of nucleotide sequence I are fluoro-modified nucleotides. and the remaining nucleotides in the sense strand of the siRNA are methoxy-modified nucleotides. and the 5'-end to the 3'-end of the antisense strand of the siRNA. The nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II in The nucleotides at the remaining positions of the antisense strand of the siRNA are ORO-modified nucleotides. is a methoxy modified nucleotide, Alternatively, the nucleotide sequence of the sense strand of the siRNA from the 5' end to the 3' end may be The nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I are fluoro-modified nucleotides. and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides. Also, from the 5' end to the 3' end, the nucleotide sequence in the antisense strand of the siRNA is The nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluoro-modified nucleotides. The remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides. It is a nucleotide, Alternatively, the nucleotide sequence of the sense strand of the siRNA from the 5' end to the 3' end may be The nucleotides at positions 7, 8 and 9 of nucleotide sequence I are fluoro-modified nucleotides; The nucleotides at the remaining positions of the sense strand of the iRNA are methoxy-modified nucleotides, In addition, the nucleotide sequence of the antisense strand of the siRNA from the 5' end to the 3' end The nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II are fluoro-modified nucleotides. and the remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides. This is the punch line.

[0243] In some embodiments, the siRNA provided by the present disclosure is as set forth in Tables 1a to 1l. siXOa1-M1, siXOa1-M2, siXOa1-M3, and siXOa1-M4, which are described in XOa2-M1, siXOa2-M2, siXOa2-M3, siXOb1-M1, si XOb1-M2, siXOb1-M3, siXOb2-M1, siXOb2-M2, si XOb2-M3, siXOc1-M1, siXOc1-M2, siXOc1-M3, si XOc2-M1, siXOc2-M2, siXOc2-M3, siXOd1-M1, si XOd1-M2, siXOd1-M3, siXOd2-M1, siXOd2-M2, si XOd2-M3, siXOe1-M1, siXOe1-M2, siXOe1-M3, si XOe2-M1, siXOe2-M2, siXOe2-M3, siXOf1-M1, si XOf1-M2, siXOf1-M3, siXOf2-M1, siXOf2-M2, si XOf2-M3, siXOg1-M1, siXOg1-M2, siXOg1-M3, si XOg2-M1, siXOg2-M2, siXOg2-M3, siXOh1-M1, si XOh1-M2, siXOh1-M3, siXOh2-M1, siXOh2-M2, si XOh2-M3, siXOi1-M1, siXOi1-M2, siXOi1-M3, si XOi2-M1, siXOi2-M2, siXOi2-M3, siXOj1-M1, si XOj1-M2, siXOj1-M3, siXOj2-M1, siXOj2-M2, si XOj2-M3, siXOk1-M1, siXOk1-M2, siXOk1-M3, si XOk2-M1, siXOk2-M2, siXOk2-M3, siXOl1-M1, si XOl1-M2, siXOl1-M3, siXOl2-M1, siXOl2-M2 and s iXOl2-M3, any one of them.

[0244] The siRNA having the above modification is not only low in cost, but also resistant to ribonucleases in the blood It is possible to make nucleic acids less susceptible to cleavage, thereby improving the stability of the nucleic acid and reducing the nucleic acid fragmentation. The modified siRNA has the property of being resistant to hydrolysis by enzymes. High mRNA suppression activity.

[0245] In some embodiments, the sense and anti-strands of the siRNA provided by the present disclosure In the sense strand, at least one single strand has a small number of phosphate ester groups in its phosphate-sugar backbone. At least some of the phosphate groups have a modifying group. The phosphate ester group having a modifying group is a phosphodiester bond in the phosphate ester group. At least one oxygen atom of the thiophosphate group is replaced with a sulfur atom. In some embodiments, the phosphate ester group having the modifying group is represented by formula (1): It is a thiophosphate group having the structure.

[0246] [ka]

[0247] These modifications stabilize the double-stranded structure of siRNA and enhance the specificity of base pairing. and maintain a high affinity.

[0248] In some embodiments, in the siRNA provided by the present disclosure, thiophosphoryl The acid ester group is 2′-nucleotide with the first nucleotide at either end of the sense or antisense strand. between the 1st nucleotide and the 2nd nucleotide at either end of the sense or antisense strand, between the nucleotide and the third nucleotide, or any combination thereof In some embodiments, the thiophosphate is present in a carboxylic acid moiety. Terminator groups are present attached at all of the above positions except the 5' end of the sense strand. In embodiments, the thiophosphate group is at all of the above positions except the 3' end of the sense strand. In some embodiments, the thiophosphate group is bonded to is present bound at at least one of the 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, 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 Between, and The second and third nucleotides from the 3' end of the antisense strand Between.

[0249] In some embodiments, the siRNA provided by the present disclosure is as set forth in Tables 1a to 1l. siXOa1-M1S, siXOa1-M2S, and siXOa1-M3S, which are described in , siXOa2-M1S, siXOa2-M2S, siXOa2-M3S, siXOb1 -M1S, siXOb1-M2S, siXOb1-M3S, siXOb2-M1S, si XOb2-M2S, siXOb2-M3S, siXOc1-M1S, siXOc1-M2 S, siXOc1-M3S, siXOc2-M1S, siXOc2-M2S, siXOc 2-M3S, siXOd1-M1S, siXOd1-M2S, siXOd1-M3S, s iXOd2-M1S, siXOd2-M2S, siXOd2-M3S, siXOe1-M 1S, siXOe1-M2S, siXOe1-M3S, siXOe2-M1S, siXO e2-M2S, siXOe2-M3S, siXOf1-M1S, siXOf1-M2S, siXOf1-M3S, siXOf2-M1S, siXOf2-M2S, siXOf2- M3S, siXOg1-M1S, siXOg1-M2S, siXOg1-M3S, siX Og2-M1S, siXOg2-M2S, siXOg2-M3S, siXOh1-M1S , siXOh1-M2S, siXOh1-M3S, siXOh2-M1S, siXOh2 -M2S, siXOh2-M3S, XOi1-M1S, siXOi1-M2S, siXO i1-M3S, siXOi2-M1S, siXOi2-M2S, siXOi2-M3S, siXOj1-M1S, siXOj1-M2S, siXOj1-M3S, siXOj2- M1S, siXOj2-M2S, siXOj2-M3S, siXOk1-M1S, siX Ok1-M2S, siXOk1-M3S, siXOk2-M1S, siXOk2-M2S , siXOk2-M3S, siXOl1-M1S, siXOl1-M2S, siXOl1 -M3S, siXOl2-M1S, siXOl2-M2 and siXOl2-M3S There is one.

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

[0251] The conventional 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide is As known to those of skill in the art, for example, a 5'-phosphate nucleotide may have the following structure: .

[0252] [ka]

[0253] See, for example, Anastasia Khvorova and Jonathan K. Watts,The chemical evolution of oligo nucleotide therapies of clinical utility . Nature Biotechnology,2017,35(3): 238~4 8 discloses the following four types of 5'-phosphate analog modified nucleotides:

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

[0255] In some embodiments, the 5'-phosphate nucleotide is a 5'-phosphate nucleotide as shown in formula (2): A 5'-phosphate analog modified nucleotide is a nucleotide that contains a 5'-phosphate modification. Vinyl phosphate ester (5'-(E)-vinylphospho) represented by formula (3) nucleotide containing a thiol ester (E-VP) modification, or a nucleotide having the formula (5) It is a phosphate modified nucleotide.

[0256] In some embodiments, the siRNA provided by the present disclosure is as set forth in Tables 1a to 1l. siXOa1-M1P1, siXOa1-M2P1, and siXOa1-M 3P1, siXOa2-M1P1, siXOa2-M2P1, siXOa2-M3P1, siXOa1-M1SP1, siXOa1-M2SP1, siXOa1-M3SP1, s iXOa2-M1SP1, siXOa2-M2SP1, siXOa2-M3SP1, si XOb1-M1P1, siXOb1-M2P1, siXOb1-M3P1, siXOb2 -M1P1, siXOb2-M2P1, siXOb2-M3P1, siXOb1-M1S P1, siXOb1-M2SP1, siXOb1-M3SP1, siXOb2-M1SP 1, siXOb2-M2SP1, siXOb2-M3SP1, siXOc1-M1P1, siXOc1-M2P1, siXOc1-M3P1, siXOc2-M1P1, siXO c2-M2P1, siXOc2-M3P1, siXOc1-M1SP1, siXOc1- M2SP1, siXOc1-M3SP1, siXOc2-M1SP1, siXOc2-M 2SP1, siXOc2-M3SP1, siXOd1-M1P1, siXOd1-M2P 1, siXOd1-M3P1, siXOd2-M1P1, siXOd2-M2P1, si XOd2-M3P1, siXOd1-M1SP1, siXOd1-M2SP1, siXO d1-M3SP1, siXOd2-M1SP1, siXOd2-M2SP1, siXOd 2-M3SP1, siXOe1-M1P1, siXOe1-M2P1, siXOe1-M 3P1, siXOe2-M1P1, siXOe2-M2P1, siXOe2-M3P1, siXOe1-M1SP1, siXOe1-M2SP1, siXOe1-M3SP1, s iXOe2-M1SP1、siXOe2-M2SP1、siXOe2-M3SP1、si XOf1-M1P1、siXOf1-M2P1、siXOf1-M3P1、siXOf2 -M1P1、siXOf2-M2P1、siXOf2-M3P1、siXOf1-M1S P1、siXOf1-M2SP1、siXOf1-M3SP1、siXOf2-M1SP 1、siXOf2-M2SP1、siXOf2-M3SP1、siXOg1-M1P1、 siXOg1-M2P1、siXOg1-M3P1、siXOg2-M1P1、siXO g2-M2P1、siXOg2-M3P1、siXOg1-M1SP1、siXOg1- M2SP1、siXOg1-M3SP1、siXOg2-M1SP1、siXOg2-M 2SP1、siXOg2-M3SP1、siXOh1-M1P1、siXOh1-M2P 1、siXOh1-M3P1、siXOh2-M1P1、siXOh2-M2P1、si XOh2-M3P1、siXOh1-M1SP1、siXOh1-M2SP1、siXO h1-M3SP1、siXOh2-M1SP1、siXOh2-M2SP1、siXOh 2-M3SP1、XOi1-M1P1、siXOi1-M2P1、siXOi1-M3P 1、siXOi2-M1P1、siXOi2-M2P1、siXOi2-M3P1、si XOi1-M1SP1、siXOi1-M2SP1、siXOi1-M3SP1、siX Oi2-M1SP1、siXOi2-M2SP1、siXOi2-M3SP1、siXO j1-M1P1、siXOj1-M2P1、siXOj1-M3P1、siXOj2-M 1P1、siXOj2-M2P1、siXOj2-M3P1、siXOk1-M1P1、 siXOk1-M2P1, siXOk1-M3P1, siXOk2-M1P1, siXO k2-M2P1, siXOk2-M3P1, siXOl1-M1P1, siXOl1-M 2P1, siXOl1-M3P1, siXOl2-M1P1, siXOl2-M2P1, siXOl2-M3P1, siXOj1-M1SP1, siXOj1-M2SP1, si XOj1-M3SP1, siXOj2-M1SP1, siXOj2-M2SP1, siX Oj2-M3SP1, siXOk1-M1SP1, siXOk1-M2SP1, siXO k1-M3SP1, siXOk2-M1SP1, siXOk2-M2SP1, siXOk 2-M3SP1, siXOl1-M1SP1, siXOl1-M2SP1, siXOl1 -M3SP1, siXOl2-M1SP1, siXOl2-M2SP1 and siXOl2 -M3SP1 or one of the following:

[0257] The inventors of the present disclosure have found that the siRNA provided by the present disclosure is plasma and lysosome stable. We unexpectedly found that not only was the activity significantly improved, but the activity of suppressing the target mRNA was also high. Ta.

[0258] The siRNA provided by the present disclosure can be prepared by a conventional siRNA preparation method in the art (e.g. For example, solid-phase synthesis and liquid-phase synthesis. Commercial customization services are already available. The use of mers to introduce modified nucleotide groups into the siRNAs described in this disclosure and methods for preparing nucleoside monomers having corresponding modifications, and modified nucleoside monomers. Methods for introducing leutide groups into siRNA are also well known to those skilled in the art.

[0259] <Drug Composition> The present disclosure relates to a drug comprising the above-mentioned siRNA as an active ingredient and a pharma- ceutically acceptable carrier. A composition is provided.

[0260] The pharma- ceutical acceptable carrier may be a carrier commonly used in the field of siRNA administration. Often, for example, magnetic nanoparticles (e.g. , nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes (carbon nanotubes, mesoporous silicon n), calcium phosphate nanoparticles rticles), polyethyleneimine (PEI) , polyamidoamine dendrimer (polyamidoamine (PAMAM) d endrimer), poly(L-lysine, PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane ( 1,2-dioleoyl-3-trimethylammonium-propane , DOTAP), poly(D- or L-lactic acid / glycolic acid copolymer (poly(D&L-lactide / glycolide copolymer) (actic / glycolic acid) copolymer, PLGA), poly(acetylacetonate) Poly(2-aminoethyl ethylene phosphate) Poly(N,N-dimethylaminoethylene phosphate), PPEEA) and poly(N,N-dimethylaminoethylene Poly(2-dimethylaminoethyl methacrylate) acrylate), PDMAEMA) and one or more of their derivatives. The present invention is not limited to these.

[0261] The content of siRNA and a pharma- ceutical acceptable carrier in the pharmaceutical composition is particularly However, there is no requirement that the amount of each component be in the range of 100-150% by weight, and the amount of each component may be in the range of 100-150% by weight. The weight ratio of the siRNA to the pharma- ceutically acceptable carrier may be 1:(1 to 500). In some embodiments, the weight ratio is 1:(1 to 50).

[0262] In some embodiments, the drug composition may contain other pharma- ceutically acceptable excipients. The additive may be one or more of various agents or compounds commonly used in the art. For example, the pharma- ceutically acceptable other additives may be a pH buffer, At least one of a protective agent and an osmolality regulator may be included.

[0263] The pH buffer solution is tris(hydroxymethylaminomethane hydrochloride) having a pH of 7.5 to 8.5. Buffer solution (tris(hydroxymethyl) aminomethane hyd rochloride buffer) and / or phosphate buffer of pH 5.5 to 8.5 For example, it may be a phosphate buffer solution having a pH of 5.5 to 8.5.

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

[0265] The osmolality adjusting agent may be sodium chloride and / or potassium chloride. The content of the osmolality adjusting agent is such that the osmolality of the drug composition is 200 to 700 mOsm / kg. Depending on the desired osmolality, one skilled in the art can determine the osmolality of the The content of the osmotic regulator can be easily determined.

[0266] In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injectable solution. Alternatively, the lyophilized powder may be mixed with a liquid additive at the time of administration to form a liquid preparation. The liquid formulation may be administered by subcutaneous, intramuscular or intravenous injection, but is not limited thereto. First, administer the drug to the lungs by aerosol, or deliver the drug to other organ tissues (e.g., the liver) through the lungs by aerosol. In some embodiments, the administration may be to, but is not limited to, The pharmaceutical composition is adapted for intravenous administration.

[0267] In some embodiments, the drug composition may be in the form of a liposomal formulation. In some embodiments, the pharma- ceutically acceptable carrier for use in the liposomal formulation is The carrier is an amine-containing transfection compound (hereinafter also referred to as an organic amine), a co-lipid, The organic amines include, for example, organic amino acids and / or PEGylated (PEGylated) lipids. The lipids, co-lipids and PEGylated lipids were prepared as described in CN103380113A (the entirety of which is incorporated herein by reference). The amine-containing transfection compounds described herein or their pharmaceutical compositions are or one or more selected from the group consisting of a salt or derivative thereof which is acceptable for There may be multiple types.

[0268] In some embodiments, the organic amine is a compound described in CN103380113A. The compound may be a compound represented by formula (201) 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 water. or C1-C 20 is a hydrocarbon chain, Each Y 101 or Z 101 are each independently C=O, C=S, S=O, CH-OH or SO2, Each R 101 , R 102 , R 103 , R 104 , R 105 , R 106 or R 107 That each independently represents hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a ring or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic groups of the formula , branched or straight chain acyl groups, substituted or unsubstituted branched or straight chain aryl groups, substituted or unsubstituted a substituted, branched or straight chain heteroaryl group; x is an integer from 1 to 10; n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1, If m=p=0, R 102 is hydrogen, When at least one of n or m is 2, R 103 and nitrogen in formula (201) , forming 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, and "HCC" represents a hydrocarbon chain. represents 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) 10 1 and R 102 Each of may independently be an optionally substituted or unsubstituted branched or linear alkyl group. alkyl or alkenyl, said alkyl or alkenyl having from 3 to about 20 carbon atoms; For example, 8 to about 18 carbon atoms and 0 to 4 double bonds, for example, 0 to 2 double bonds. It has a combination.

[0272] In some embodiments, when n and m are each independently 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. where each "HCC" represents a hydrocarbon chain and each * is R 103 and nitrogen in formula (201) Indicates possible bond points with the atom, and any * Each H on the position corresponds to a nitrogen atom in formula (201). It may be substituted for binding.

[0274] The compound of formula (201) was prepared according to the method described in CN103380113A. Good too.

[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 may be cholesterol, a cholesterol analogue, and / or a cholesterol analogue. It is a derivative of The PEGylated lipid is 1,2-dipalmitamido-sn-glycero-3-phosphatidylcholine. Triethanolamine-N-[methoxy(polyethylene glycol)]-2000(1,2 -dipalmitoyl-sn-glycero-3-phosphatidylet hanolamine-N-[methoxy(polyethylene glyco l)]-2000).

[0278] In some embodiments, the pharmaceutical composition further comprises the organic amine, the auxiliary lipid, The molar ratio of the PEGylated lipids was (19.7-80):(19.7-80):(0.3 For example, it may be (50-70):(20-40):(3-20). stomach.

[0279] In some embodiments, the siRNA of the present disclosure and the amine-containing transfection The drug composition particles formed by the injection reagent have an average diameter of about 30 nm to about 200 nm. Generally, the average diameter of the liposome particles is about 40 nm to about 135 nm, and more generally, The diameter is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or The average diameter of the liposome particles is about 70 nm to about 90 nm. For example, the average diameter of the liposome particles is about 30, 40 0, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 1 40, 150 or 160 nm.

[0280] In some embodiments, the siRNA of the present disclosure and the amine-containing transfection In the drug composition formed by the coupling reagent, siRNA and total lipid (e.g., organic amine , auxiliary lipid and / or PEGylated lipid) weight ratio (weight / weight ratio) is about 1:1 to about 1:5 0, approx. 1:1~approx. 1:30, approx. 1:3~approx. 1:20, approx. 1:4~approx. 1:18, approx. 1:5~ 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 For example, the weight ratio of siRNA to total lipids of the present disclosure is in the range of about 1:6 to about 1:10. Approximately 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 is marketed with each component present independently. In some embodiments, the present disclosure provides a liquid formulation. and a pharma- ceutical composition comprising the siRNA provided by may be prepared according to various known methods, and may be used according to the present disclosure instead of conventional siRNA. In some embodiments, the siRNA provided by the following method may be used. It may also be prepared as follows.

[0282] The organic amine, auxiliary lipid, and PEGylated lipid were suspended in alcohol at the above molar ratio and mixed uniformly. The amount of alcohol is adjusted so that the total mass concentration of the resulting lipid solution is 2 to 25%. The alcohol is determined to be, for example, 8 to 18 mg / mL. Pharmaceutically acceptable alcohols, such as ethanol, propylene glycol, benzyl Alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 30 0, polyethylene glycol 400, etc., are selected from alcohols that are liquid at around room temperature. The alcohol may be one or more of the above alcohols, and may be, for example, ethanol.

[0283] The siRNA provided by the present disclosure is dissolved in a buffered salt solution to obtain an aqueous siRNA 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 solution is preferably such that the siRNA concentration is 0.6 mg / mL or less, e.g., 0.2 to 100 mg / mL. The buffer salts are soluble acetate, soluble citrate, and soluble sucrose. One or more selected from the group consisting of sodium acetate and / or potassium acetate. It may be Umu.

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

[0285] The cultured liposome preparation is concentrated or diluted, impurities are removed, sterilized, and the liposome preparation is prepared according to the present disclosure. The pharmaceutical composition is obtained, the physicochemical parameters of which are pH 6.5-8; The encapsulation efficiency is 80% or more, the particle size is 40-200 nm, and the polydispersity index is 0.30. The osmolality is 250-400 mOsm / kg or less. For example, The pH is 7.2 to 7.6, the entrapment efficiency is 90% or more, and the particle size is 60 to 100 nm. The polydispersity index may be 0.20 or less, and the osmotic pressure may be 300 to 400 mOsm / kg.

[0286] Here, concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. Any of various conventional methods may be employed as the method for removing impurities. For example, using a tangential flow system and a hollow fiber column, ultrafiltration may be performed under conditions of 100 KDa, and the ultrafiltration exchange solution may be a phosphate buffer (PBS) at pH 7.4. As the sterilization method, any of 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 including the above siRNA and a complex group that is complexed and bound to the siRNA.

[0288] Generally, the complex group includes at least one target group that is pharmaceutically acceptable 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, may be at the 5' end of the antisense strand, or may be in the internal sequence of the siRNA. In some embodiments, the complex site between the siRNA and the complex group is at the 3' end of the sense strand of the siRNA.

[0289] ​​​​​In some embodiments, the conjugated group is a phosphate group of a nucleotide, a 2'-hydroxyl group In some embodiments, the conjugated group may be linked to a hydroxyl group or a base. It may be linked to the 3'-hydroxy group, in which case the internucleotide is a 2'-5' linker. The conjugated groups are attached to the ends of the siRNA strands via phosphate diester bonds. When the nucleotide is linked to the phosphate group of a nucleotide, it is usually linked to the internal sequence of the siRNA. The various methods of attachment are described in Muthia et al. h Manoharan et.al. siRNA conjugates carr ying sequentially assembled trivalent N- acetylgalactosamine linked through nucleus osides elicit robust gene silencing in v ivo in hepatocytes. ACS Chemical biology ,2015,10 (5): 1181~7.

[0290] In some embodiments, the bond between the siRNA and the conjugated group is acid labile or reducible. These chemical bonds can be linked to the endosomal acidic environment of the cell. The siRNA can be released by decomposition. The conjugated group was bound to the sense strand of siRNA, and the effect of conjugation on siRNA activity was examined. can be reduced as much as possible.

[0291] In some embodiments, the pharma- ceutically acceptable targeting group is a pharma- ceutically acceptable targeting group that is suitable for use in the field of siRNA administration. Ligands commonly used in the art, such as the 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 is cholesterol, bile lipophilic molecules such as juices, vitamins (e.g., tocopherols), and lipid molecules of different chain lengths; Polymers such as polyethylene glycol, polypeptides such as membrane-permeable peptides, and aptamers mers, antibodies, quantum dots, lactose, polylactose, mannose, galactose It contains sugars such as galactosamine (GalNAc) and folic acid (folate). , asialoglycoprotein, asialoglycoprotein residues, lipoproteins (e.g., high density lipoprotein , low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth Target molecules such as receptor ligands expressed in hepatic parenchymal cells, such as transferrin, or The ligand may be one or more selected from the ligands formed by derivatives.

[0293] In some embodiments, each of the ligands is a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is independently selected from the group consisting of is a ligand capable of binding to a receptor on the surface of liver cells. At least one of the ligands is capable of binding to a receptor on the surface of mammalian hepatocytes. In some embodiments, the at least one ligand is a receptor on the surface of human hepatocytes. In some embodiments, at least one ligand is capable of binding to is a ligand that can bind to the asialoglycoprotein receptor (ASGPR) on the liver surface. These types of ligands are known to those skilled in the art and generally act to bind to target cell surfaces. It binds to specific receptors on the surface and mediates the delivery of siRNA bound to the ligand to target cells. do.

[0294] In some embodiments, the pharma- ceutically acceptable targeting group is a target of a mammalian hepatocyte surface. Any one of the ligands that bind to the asialoglycoprotein receptor (ASGPR) on the surface In some embodiments, each ligand may be independently an asialoglycoprotein. Proteins, e.g. asialoorosomucoid (A SOR 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 ligand is a sugar. Another ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide or sugar derivative. In an embodiment, at least one of the ligands may be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. Each ligand may be independently a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or In some embodiments, each or at least one of the ligands is selected from the group consisting of monosaccharide derivatives. At least one of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives conductors, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives The glycerol derivative is selected from the group consisting of glycerol, glycerol, fructose and its derivatives, and sialic acid.

[0296] In some embodiments, each of the ligands is D-mannopyranose, L-mannopyranose, Pyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose Glucose, L-glucose, D-galactose, L-galactose, α-D-mannose D-mannose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranolate α-D-glucopyranose, β-D-glucopyranose, α-D-glucofurano β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranolate α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose Ranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N -Acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionyl Galactosamine, Nn-Butyrylgalactosamine, N-Isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucose Glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-di Deoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy Oxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid , 5-thio-β-D-glucopyranose, methyl 2,3,4-tris-O-acetyl-1 -Thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranoside Ranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio O-α-D-glucoheptopyranoside, 2,5-anhydro-D-allonitrile, riboflavin from D-ribose, D-4-thioribose, L-ribose or L-4-thioribose Other options for the ligand include, for example, CN1053780. 82A, the disclosure of which is incorporated herein by reference in its entirety. Can be enjoyed.

[0297] In some embodiments, the siRNA complex includes a pharma- ceutically acceptable target. The target group may be galactose or N-acetylgalactosamine, and may be galactose or The N-acetylgalactosamine molecule may be monovalent, divalent, trivalent or tetravalent. The monovalent, divalent, trivalent, and tetravalent groups are composed of siRNA molecules and galactose as a targeting group, respectively. The siRNA complex formed from the conjugate group containing galactosamine or N-acetylgalactosamine molecules The molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in It should be understood to mean 1:1, 1:2, 1:3 or 1:4. In an embodiment, the pharma- ceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, the siRNA described herein is N-acetylglucosamine. When conjugated to a conjugated group containing an amine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, the siRNA described in the present disclosure is N-acetylglucosamine. When conjugated to a galactosamine-containing conjugating group, the N-acetylgalactosamine molecule is trivalent.

[0298] The targeting group may be attached to the siRNA molecule via a suitable linker, and those skilled in the art will appreciate that the targeting group may be linked to the siRNA molecule via a suitable linker. An appropriate linker can be selected depending on the specific type of the target group. For the types of targeting groups and methods of binding to siRNA, see WO2015006740A2. Reference may be made to the disclosure of which, 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 containing an amide bond having a structure represented by formula (302), L A has one of the target groups and one of the L C Bonded to the part by ether bond will be done.

[0301] [ka] L B is a chain portion containing N-acylpyrrolidine having the structure shown in formula (303). The chain portion has a carbonyl group at one end thereof, and the L C Bonded to the part by an amide bond and has an oxygen group at the other end, and is bound to the siRNA via a phosphate ester bond.

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

[0303] In some embodiments, n=3 and L C Trihydroxymethylaminomethyl In the case of a tetravalent linker group based on a phenyl group, -(L A ) 3 Trihydroxy Methylaminomethane-L B - N-acetylgalactosamine and siRNA molecules The structure of the siRNA complex formed by binding is shown in the following formula (304).

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

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

[0306] In some embodiments, the 3' end of the sense strand of the siRNA described herein is , linker-(L A ) 3 Trihydroxymethylaminomethane-L B -Three N-A Covalently conjugated to a cetylgalactosamine (GalNAc) molecule, The molar ratio of GalNAc molecules is 1:3, and the structure is shown in the following formula (305): An RNA complex (hereinafter also referred to as (GalNAc)3-siRNA) is obtained.

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

[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 formula (30 7) has the structure shown in

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

[0312] The siRNA complexes were synthesized by methods already described in detail in the prior art. For example, WO2015006740A2 describes a method for preparing a complex containing multiple siRNAs. The siR of the present disclosure can be prepared by methods well known to those skilled in the art. For example, WO2014025805A1 describes the compound shown in formula (305): A method for preparing the structure has been described, and Rajeev et al. 15, 16, 903-908, a method for preparing the structure shown in formula (307) was described.

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

[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 C1-C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 a alkoxy groups; R3 is a group having the structure shown in formula A59.

[0315] [ka] wherein E1 is OH, SH, or BH2, and Nu is an siRNA of the disclosure.

[0316] R2 is a linear alkylene group of 1 to 20 carbon atoms in length, where one or more carbon Atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene Group , C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocycli olefin group and C5-C 10 one or more selected from the group consisting of heteroarylene groups; and R2 is C1-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl groups, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C1 0 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphe Nyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halo -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1 -C 10 Alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 (alkyl group) , N(C1-C 10 Alkyl group)(C1-C 10 alkylphenyl group), NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 alkyl group), -CON(C1-C 10 Alkyl group)(C1-C10 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl group)C(O )(C1-C 10 alkyl group), -N(C1-C 10 alkyl group)C(O)(phenyl group ), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylphenyl groups, -C(O)C1-C 10 Haloalkyl group, -OC(O)C1-C 10 Alkyl group, -SO 2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halo -SO2NH2, -SO2NH(C1-C 10 alkyl group), -S O2NH (phenyl group), -NHSO2(C1-C 10 Alkyl group), -NHSO2( phenyl group) and -NHSO2(C1-C 10 any of the group consisting of halogenated alkyl groups or optionally bearing one or more substituents, Each L1 is a linear alkylene group of 1 to 70 carbon atoms in length, where one or more carbon atoms Elementary atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterothic Rylene group and C5-C 10 heteroarylene groups, L1 is replaced by C1-C 10 Alkyl groups, C6-C10 Aryl groups, C5-C1 0 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group , -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylph Phenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups rogene substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C 1-C 10 Alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 Alkyl group ), N(C1-C 10 Alkyl group)(C1-C 10 alkylphenyl group), NH(C1- C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1- C 10 alkyl group), -CON(C1-C 10 Alkyl group)(C1-C 10 (alkyl group) , -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C1 0 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl group)C( O)(C1-C 10 alkyl group), -N(C1-C 10 alkyl group)C(O)(phenyl group), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylphenyl group , -C(O)C1-C 10 Haloalkyl group, -OC(O)C1-C 10 Alkyl group, -S O2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Ha halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), - SO2NH (phenyl group), -NHSO2(C1-C 10 Alkyl group), -NHSO2( Phenyl group) and -NHSO2(C1-C 10 Any of the group consisting of halogenated alkyl groups Each of these may optionally have one or more substituents.

[0317] In some embodiments, L1 is selected from the group A1 through A26, or any combination thereof. 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 C1-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 C1-C 10 is an alkyl group, JPEG2025038101000024.jpg12124 represents the site to which the group is covalently attached.

[0320] For convenience, L1 is defined as a linear alkylene group, but may also include, for example, the above-mentioned substitutions and / or Amines and alkenyl groups resulting from substitution that may not be linear or may have different names For purposes of this disclosure, the length of L1 is determined by dividing the length of the is the number of atoms in the chain that connects the bond points. A ring obtained by replacing a nitrogen atom (e.g., heterocyclylene or heteroarylene) is formed by atoms.

[0321] M1 represents a targeting group, and its definition and selectable range are the same as those of the targeting group described above. In some embodiments, each M1 is an asialoglycoprotein on the surface of a mammalian hepatocyte. It is one independently selected from ligands that have affinity for the receptor.

[0322] M1 has affinity for the asialoglycoprotein receptor on the surface of mammalian hepatocytes. In some embodiments, n1 is an integer from 1 to 3. n3 may be an integer of 0 to 4; and the M1 target in the siRNA complex may be Ensure that the number of groups is at least 2. In some embodiments, n1+n 3≧2, whereby the number of M1 targeting groups is at least 3 and the number of M1 targeting groups and the liver surface and the siRNA complex more readily binds to the asialoglycoprotein receptor on the surface of the Facilitating uptake into cells through endocytosis (intracellular uptake) As can be seen from the experiment, when the number of M1 targeting groups is three or more, The increase in the ease of binding to the asialoglycoprotein receptor on the liver surface is unclear. Considering various aspects such as ease of synthesis, structure / process cost, and delivery efficiency, In some embodiments, n1 is an integer from 1 to 2, and n3 is an integer from 0 to 1; And n1+n3=2~3.

[0323] In some embodiments, m1, m2, or m3 is independently selected from an integer of 2 to 10. When the M1 targeting group is selected, the spatial position between the M1 targeting group and the asialoglycoprotein on the liver surface is determined. The siRNA conjugates provided by the present disclosure can be adapted to bind to protein receptors. In order to make the structure simpler, easier to synthesize, and / or less costly, several In this embodiment, m1, m2, and m3 are each independently an integer of 2 to 5, In some embodiments, m1=m2=m3.

[0324] R 10 , R 11 , R 12 , R 13 , R 14 or R 15 But, H, C1-C 10 Alkyl group , C1-C 10 Halogenated alkyl groups and C1-C 10 Independently from each alkoxy group When one of the above is selected, any of them can change the properties of the siRNA complex of the present disclosure. It will be understood by those skilled in the art that the objectives of the present disclosure can be achieved without the need for some In the embodiment, R 10 , R 11 , R 12 , R 13 , R 14 or R 15 are respectively In some embodiments, R1 is independently selected from H, a methyl group, or an ethyl group. 0, R 11 , R 12 , R 13 , R 14 and R 15are both H.

[0325] R3 is a group having the structure shown in formula A59, where E1 is OH, SH, or BH2. In view of the availability of preparation raw materials, in some embodiments, E1 is OH or It's SH.

[0326] R2 is selected to achieve a bond between the N atom on the nitrogen-containing backbone and A59. In the context of the Detailed Description, "nitrogen-containing backbone" means R 10 , R 11 , R 12 , R 13 , R 14 and R 15 It refers to a chain structure in which a carbon atom to which is bonded and a N atom are bonded to each other. Thus, R2 can be used to attach the A59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, the linker group is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, In preparing the siRNA complex of formula (308), the R2 group is preferably a nitrogen-containing backbone. The site bonded to the N atom in the upper position and the site bonded to the P atom in R3 are both included. In some embodiments, the N atom on the nitrogen-containing backbone in R2 must be The moiety bonded to the P atom on R3 forms an amide bond with the N atom. In some embodiments, R2 is a phosphate ester bond with the P atom. It may be B5, B6, B5' or B6'.

[0327] [ka] During the ceremony, JPEG2025038101000026.jpg12136 represents the site to which the group is covalently attached.

[0328] The value of q2 may range from 1 to 10, and in some embodiments, q 2 is an integer from 1 to 5.

[0329] L1 binds the M1 targeting group to the N on the nitrogen-containing backbone, forming a siRNP molecule as shown in formula (308). In some embodiments, the L 1 is a combination of one or more bonds selected from the groups of formulae A1 to A26. In an embodiment, L1 is selected from A1, A4, A5, A6, A8, A10, A11 and A13. In some embodiments, L is a combination of one or more bonds selected from the following: A combination of at least two bonds selected from A1, A4, A8, A10 and A11. In some embodiments, L1 is at least one selected from A1, A8, and A10. Both are combinations of two bonds.

[0330] In some embodiments, the length of L1 is 3 to 25 atoms, 3 to 20 atoms, It may be 4 to 15 atoms or 5 to 12 atoms. The length of L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 pieces, 14 pieces, 15 pieces, 16 pieces, 17 pieces, 18 pieces, 19 pieces, 20 pieces, 21 pieces, 22 pieces, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 It is an atom.

[0331] In some embodiments, j1 is an integer from 2 to 10. In some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10. In some embodiments, j2 is an integer from 3 to 5. R' is a C1-C4 alkyl group. In some embodiments, R' is an alkyl group, and in some embodiments, R' is a methyl group, an ethyl group, and an isopropyl group. Ra is one of A27, A28, A29, A30 and A31. In some embodiments, Ra is A27 or A28. Rb is C1-C 5 alkyl group, and in some embodiments, Rb is a methyl group, an ethyl group, an isopropyl group, In some embodiments, the aryl group is one of a propyl group and a butyl group. By selecting j1, j2, R', Ra, and Rb, respectively, the M1 target group and the nitrogen The binding to the N atom on the containing skeleton is realized, and the spatial position between the M1 targeting group and the liver surface is adjusted. It is further adapted for binding to the asialoglycoprotein receptor.

[0332] In some embodiments, the siRNA complex has the formula (403), (404), (405), (406), (407), (408), (409), (410), (411 ), (412), (413), (414), (415), (416), (417), (4 18), (419), (420), (421) or (422).

[0333] [ka] JPEG2025038101000028.jpg205132JPEG2025038101000029.jpg206131JPEG2025038101000030.jpg194120 JPEG2025038101000031.jpg233118JPEG2025038101000032.jpg214115JPEG2025038101000033.jpg162121

[0334] In some embodiments, the P atom in formula A59 is any atom in the siRNA sequence. For example, the P atom in formula A59 may be bonded to any available position of the siRNA. It may be bound to one nucleotide of either the sense strand or the antisense strand, and any number of In some embodiments, the P atom in formula A59 is any one of the P atoms in the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is linked to one nucleotide. is attached to the end of the sense strand or antisense strand of the siRNA, and in some embodiments In the formula A59, the P atom is bound to the end of the sense strand of the siRNA. The portion is the first four nucleotides from one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula A59 is a nucleotide of the siRNA. and attached to the end of the sense strand or the antisense strand, in some embodiments, The P atom in is attached to the 3' end of the sense strand of the siRNA. When the siRNA complex represented by formula (308) is bound to the above position, the siRNA complex represented by formula (308) enters the cell. When the siRNA is unwound after incorporation, it releases the single antisense strand of the siRNA and binds to the XO It is possible to block the process of mRNA translation into protein and suppress XO gene expression. .

[0335] In some embodiments, the P atom in formula A59 is a nucleotide in the siRNA. Any possible position on the nucleotide, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, It may be attached to the 3' position of the nucleotide or to the base of the nucleotide. In the formula A59, the P atom forms a phosphodiester bond to It may be attached to the 2', 3' or 5' position of the nucleotide in the siRNA. In some embodiments, the P atom in formula A59 is at the 3' end of the sense strand of the siRNA. The 3' hydroxyl group of the nucleotide is dehydrogenated to the oxygen atom (A59 is The P atom in the nucleotide sequence can be regarded as the P atom of the phosphate group contained in the siRNA. The P atom in formula A59 is the 2 nucleotide of one nucleotide in the sense strand of the siRNA. or is attached to the nucleotide by replacing the hydrogen in the '-hydroxy group, The P atom at 59 is the 5' hydroxyl group of the 5' terminal nucleotide of the sense strand of the siRNA. It is attached to the nucleotide by replacing a hydrogen in the group.

[0336] The inventors of the present disclosure have found that the siRNA complex of the present disclosure has significantly improved stability in plasma and off- Reduced target effect and high XO mRNA silencing activity It has been surprisingly found that the siRNA of the present disclosure is The siRNA may be one of the siRNAs shown in Tables 1a to 1l. The siRNA complexes 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 1l] Here, capital letters C, G, U, and A represent the base sequence of a nucleotide, and lower case letter m represents the corresponding The letter m indicates that the 1 nucleotide adjacent to the left is a methoxy-modified nucleotide. The lowercase letter f indicates that the 1 nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide. The lowercase letter s indicates that the two nucleotides on either side of the letter are thiol. P1 is bonded to the 1-membered ring adjacent to the right of P1 through a phosphate group. One nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, P1 represents a VP, Ps or represents P, and the combination letter VP is one adjacent letter to the right of the combination letter VP. Nucleotides are vinyl phosphate esters (5'-(E)-vinylphosphonates e, E-VP) modified nucleotide, and the combination letter Ps represents the combination letter One nucleotide adjacent to the right of Ps is a phosphorothioate-modified nucleotide The capital letter P indicates that the nucleotide immediately to the right of the letter P is a 5'-phosphoryl nucleotide. This indicates that the acid is a nucleotide.

[0349] In the siRNA or siRNA complex described in this disclosure, each adjacent nucleotide The bonds between the diesters are phosphate diester bonds or thiophosphate diester bonds. The non-bridging oxygen or sulfur atoms in the ester or thiophosphate bond are negatively charged. It is charged with a hydroxyl or sulfhydryl group. The hydrogen ions at the hydroxyl or sulfhydryl groups may be partially or completely substituted. may be entirely substituted with a cation. The cation may be any cation, for example a metal Cation, ammonium ion NH4 + may be one of the organic ammonium cations. In one embodiment, the cation is an alkali metal ion, in consideration of improving solubility. ions, ammonium cations formed by tertiary amines and quaternary ammonium cations The alkali metal ion is one or more selected from the group consisting of K+ And / or Na + and the cation formed by the tertiary amine may be formed by triethylamine. Ammonium ions formed by ammonium ions and / or N,N-diisopropylethylamine The ammonium ion may be formed by the siRNP described in this disclosure. A or the siRNA complex may be present at least partially as a salt. In the case of The sulfur atoms are at least partially bound to sodium ions, and the siR The NA or siRNA complexes are present as sodium salts or partial sodium salts.

[0350] As will be apparent to those skilled in the art, nucleoside monomers having corresponding modifications can be used. The modified nucleotide groups can be introduced into the siRNAs described in this disclosure by using Methods for preparing nucleoside monomers having corresponding modifications and methods for preparing modified nucleoside monomers are provided. Methods for introducing nucleotide groups into siRNA are also well known to those skilled in the art. The leioside monomers may be commercially available or may be prepared by known methods.

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

[0352] In some embodiments, the siRNA complex of formula (308) can be prepared in the following manner: The method can be carried out under the conditions of phosphoramidite solid phase synthesis. Depending on the nucleotide types and order of the sense and antisense strands of the siRNA, the 3' Nucleoside monomers are sequentially linked from the 5' to the 5', and the linkage of each nucleoside monomer is The process involves four reactions: deprotection, coupling, capping, and oxidation or sulfurization. and isolating and annealing the sense and antisense strands of the siRNA. , the siRNA of the present disclosure.

[0353] The method also includes reacting a compound of formula (32) under coupling reaction conditions and in the presence of a coupling reagent. 1) The compound shown in 1) is mixed with a nucleoside monomer or a nucleotide bound to a solid support. and contacting the compound of formula (321) with the sequence to form a nucleotide sequence. The compound shown in formula (321) below is a conjugated molecule. It is also called.

[0354] [ka] During the ceremony, R4 is capable of binding to siRNA represented by Nu in the compound represented by formula (308). In some embodiments, R4 is a si which is represented by a covalent bond to Nu. In some embodiments, R4 is a group capable of binding to RNA. A group capable of being conjugated to any functional group of the siRNA represented by Nu via a diester bond, Each S1 was independently prepared by substituting all active hydroxy groups in M1 with YCOO- groups. each Y is a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, ethyl group, trichloromethyl group, dichloromethyl group, chloromethyl group, ethyl group, n-propyl group Independent of propyl, isopropyl, phenyl, halophenyl and alkylphenyl groups 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 , The definitions and selectable ranges of L1 and M1 are as described above.

[0356] R4 provides a bond with the N atom on the nitrogen-containing backbone and is represented by the formula (308). In some embodiments, the aryl group is selected to provide a suitable reactive site for the synthesis of the A-complex. In this case, R4 is an R2 linker group or a protected R2 linker group, and It contains a functional group that can form the structure shown in A59 with siRNA.

[0357] In some embodiments, R4 is an siRNA or nucleoside molecule designated Nu. a first functional group capable of forming a phosphite ester with a group on the monomer; and a hydroxy group. or a second functional group capable of reacting with an amino group to form a covalent bond, and a solid support bound by a bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxy group or a protected hydroxy group. In an embodiment, the second functional group is a phosphoramidite, a carboxy group or a carbo In some embodiments, the second functional group is a phosphate. a solid support that is bound to another moiety of the molecule, said covalent bond being a hydroxy or amino group; In some embodiments, the solid support is formed of a phosphate ester In some embodiments, the bond is a carboxylic acid ester bond or an amide bond. In one embodiment, the solid support is a resin.

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

[0359] [ka] In the formula, q1 is an integer of 1 to 4, X is O or NH, and M + is a cation and R k is a hydroxy protecting group, SPS represents a solid phase support, JPEG2025038101000048.jpg11126 represents the site where the group is attached to the covalent moiety.

[0360] In some embodiments, the first functional group is a phosphine oxide, as shown in formula (C3): The phosphoramidite group may be at any position on the nucleotide. Coupling with a hydroxy group, for example, a hydroxy group at the 2' position or a hydroxy group at the 3' position This is reacted to form a phosphite, which is then oxidized or sulfurized to form the diphosphate shown in formula A59. The conjugated molecule is conjugated to the siRNA by forming an ester or thiophosphate bond. In this case, even if the second functional group is not present, the compound of formula (321) can be The nucleotide can be conjugated to obtain the siRNA complex shown in formula (308). In this case, siRNP is synthesized by a method such as phosphoramidite solid phase synthesis. After obtaining the sense or antisense strand of A, a compound of formula (321) and a nucleotide sequence In the latter step, the hydroxyl group on the terminal nucleotide is reacted with By forming a phosphodiester bond or a thiophosphate bond, The compound is conjugated to the 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 support. The reaction provides a composite molecule comprising a solid support. The functional group of 2 is a carboxy group, a carboxyl group, or a carboxyl group as shown in formula (C1), (C2) or (C3). The second functional group includes a carboxylate or a phosphoramidite. In the case of a compound of formula (321) containing a hydroxyl group on a solid support, e.g., a resin, The group or amino group is esterified or amidated to form a carboxylic acid ester bond. The second functional group is a phosphoramidite, forming a conjugated molecule including the solid support. When the compound of formula (321) contains a hydroxyl group, it is possible to combine the compound of formula (321) with a conventional solid support, e.g., a hydroxyl group in a resin. The hydroxyl group was then reacted with the hydroxyl group, which was then oxidized and bonded via a phosphodiester bond. Then, the solid-phase carrier-bound product is reacted with the solid-phase carrier to form a complex molecule. The nucleoside monomers are sequentially linked together according to the phosphoramidite solid phase synthesis method to form a complex. The sense or antisense strand of the siRNA is obtained with the group attached. During the phase synthesis, the first functional group is deprotected and then subjected to coupling reaction conditions. The nucleoside is coupled to a phosphoramidite group on a nucleoside monomer below.

[0362] In some embodiments, the first functional group is a hydroxy group or a protected hydroxy group. The second functional group includes an oxy group, as shown in formula (C1') or (C3'): Carboxylic acid ester bonded solid phase support or amide bonded solid phase support The solid phase carrier may be a solid support or a solid support bound by a phosphate ester bond. The compound of formula (321) was used in place of the solid support and the phosphoramidite solid phase synthesis method was followed. The nucleoside monomers are sequentially linked to form the sense or axon strand of the siRNA to which the complex group is attached. Obtain the antisense strand.

[0363] In some embodiments, the carboxylate is -COO - M + This may be expressed as So, M + is a cation, e.g., a metal cation, the ammonium cation NH + , In one embodiment, the gold cation is one selected from the group consisting of organic ammonium cations. The group ion is one selected from alkali metal ions, for example, K + Or Na + in In order to improve the solubility and facilitate the reaction, some embodiments are In this case, the organic ammonium ion is the ammonium cation formed by a tertiary amine. or quaternary ammonium cations, e.g., ammonia formed by triethylamine. Ammonium ion or ammonium ion formed by N,N-diisopropylethylamine In some embodiments, the carboxylate is triethylamine carboxylate. salt or N,N-diisopropylethylamine carboxylate.

[0364] In some embodiments, R4 is selected from the group consisting of formula (B9), (B10), (B9′), (B1 0'), (B11), (B12), (B11') or (B12') .

[0365] [ka] In the formula, q1 is an integer of 1 to 4, q2 is an integer of 1 to 10, and X is O or NH. Ri, M + is a cation and R k is a hydroxy protecting group, SPS represents a solid phase support, JPEG2025038101000050.jpg10140 represents the site at which the group is attached to the covalent linking moiety. In some embodiments, q2 is an integer from 1 to 5. In embodiments, R4 comprises a structure shown in formula (B9) or (B10). In embodiments, R4 comprises a structure shown in formula (B11) or (B12).

[0366] In some embodiments, R k Tr (trityl group), MMTr (4-methoxy trityl group), DMTr (4,4'-bismethoxytrityl group), TMTr (4,4', 4″-trimethoxytrityl group). In some embodiments, R k DMTr, i.e., 4,4'-bismethoxytrityl (4,4'-dimetho xytrityl).

[0367] The definition of L1 is as described above.

[0368] In some embodiments, L1 binds the M1 targeting group to an N atom on the nitrogen-containing backbone. , is used to provide liver targeting functionality to the siRNA complex shown in formula (308). In some embodiments, L1 includes any one or combination of A1 to A26. nothing.

[0369] From the above description, it will be readily understood by those skilled in the art that the phosphorylation Compared with the midite solid phase synthesis method, the above first functional group and the optional second functional group allow for complex The nucleotide sequence may be inserted at any possible position in the nucleotide sequence, for example at the end of the nucleotide sequence, and obtaining an siRNA complex represented by formula (308) linked to the end of the nucleotide sequence. Accordingly, unless otherwise specified, the following refers to siRNA complexes and / or complexes. In the description of the preparation of molecules, terms such as "deprotection", "coupling", "capping", When referring to reactions such as "oxidation", "sulfurization", etc., the term refers to the phosphoramidite nucleic acid reactions known in the art. It should be understood that reaction conditions and reagents relating to solid phase synthesis methods also apply to these reactions. Exemplary reaction conditions and reagents are described in detail below.

[0370] In some embodiments, each S1 is independently M1. In the formula, each S1 independently represents a hydroxy group in M1, and In some embodiments, each S1 is independently a group protected by a protecting group. All of the active hydroxy groups present in are protected with hydroxy protecting groups. In embodiments, any hydroxy protecting group known to one of skill in the art can be used to protect the active hydroxyl group in M1. In some embodiments, the protected hydroxyl group can be used to protect the hydroxyl group. The hydroxy group may be represented by the formula YCOO-, where each Y is independently C1-C 10 Al Alkyl groups and C6-C 10 aryl groups, wherein the C1-C 10 Alkyl Groups and C6-C 10 The aryl group is optionally substituted with one or more substituents, said substituents being , halogen, and a C1-C6 alkyl group. In the formula, each Y is independently a methyl group, a trifluoromethyl group, a difluoromethyl group, a monomethyl group, Monofluoromethyl group, trichloromethyl group, dichloromethyl group, chloromethyl group, ethyl group, n-propyl group, isopropyl group, phenyl group, halophenyl group and C1-C6 arsenic group alkylphenyl groups.

[0371] In some embodiments, each S1 is independently selected from the group consisting of formulae A46-A54. is selected from the group.

[0372] [ka]

[0373] In some embodiments, S1 is of formula A49 or A50.

[0374] In some embodiments, each Y is a methyl group, a trifluoromethyl group, a difluoro Methyl group, fluoromethyl group, trichloromethyl group, dichloromethyl group, chloromethyl group , ethyl group, n-propyl group, isopropyl group, phenyl group, halophenyl group and alkyl group. In some embodiments, Y is independently selected from the group consisting of phenyl and phenyl. It is a ethyl group.

[0375] As described above, the method for preparing the siRNA complex represented by formula (308) is The other strand of the siRNA is synthesized (for example, the sense strand of the siRNA to which the conjugated molecule is bound in the above step). When the siRNA is synthesized, the antisense strand of the siRNA is further synthesized according to a solid phase synthesis method. and vice versa), isolating and annealing the sense and antisense strands. Specifically, in the isolating step, the nucleotide sequence and / or The solid support bound to the conjugate molecule is cleaved and the necessary protecting groups are removed (in this case In the case of the compound of formula (321), each S1 group in the compound of formula (321) is converted to the corresponding M1 target group), The sense strand (or antisense strand) of the siRNA bound to the synthetic molecule and the corresponding antisense strand The sense strand (or antisense strand) is obtained, and the sense strand and the antisense strand are annealed to form a double-stranded RNA. A structure is formed to obtain the siRNA complex shown in formula (308).

[0376] In some embodiments, the method for preparing the siRNA complex represented by formula (308) is Under coupling reaction conditions and in the presence of a coupling reagent, a compound of formula (321) can be obtained. contacting the first nucleoside monomer at the 3' end of the sense strand or the antisense strand; The compound represented by formula (321) is attached to the first nucleotide in the sequence, and phospho The desired sense or antisense nucleotide species are synthesized under the conditions of fluoroamidite solid phase synthesis. Depending on the type and order, nucleoside monomers are linked in the 3' to 5' direction to form siRNP. A step of synthesizing a sense strand or an antisense strand of A, the compound of formula (321) being R 4, a first functional group including a protected hydroxy group, and a compound represented by formula (C1') or (C3') A compound comprising a second functional group having the structure shown in Prior to coupling with the mer, the compound of formula (321) is deprotected and each nucleoside monomer is coupled. The reaction involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. Obtaining the sense or antisense strand of the combined nucleic acid; phosphoramidite solid phase synthesis Under the conditions, the 3' to 5' sequence is determined by the type and order of nucleotides in the antisense or sense strand. Nucleoside monomers are sequentially linked toward the antisense or sense strand of the nucleic acid. Each nucleoside monomer bond is deprotected, coupled, capped, oxidized or The reaction involves four reactions: sulfurization, removal of protecting groups, cleavage from the solid support, and isolation and purification to obtain the sense strand. and obtaining and annealing an antisense strand.

[0377] In some embodiments, the method for preparing the siRNA complex represented by formula (308) is The types and order of nucleotides in the sense strand or antisense strand of the double-stranded siRNA The nucleoside monomers are linked in sequence from 3' to 5' to form the sense strand and the α strand. The antisense strand is synthesized, and the bonds of each nucleoside monomer are deprotected, coupled, and capped. The reaction involves four steps: ping, oxidation or sulfurization, and the sense strand bound to a solid support, and Obtaining the antisense strand bound to the substrate; coupling reaction conditions and coupling reagents In the presence of The antisense strand is contacted with a carrier and a first phosphoramidite group is attached to R4. A compound of formula (321) containing a functional group is attached to the sense strand or antisense strand, and a protecting group is formed. The siRNA with the complex group bound thereto is then cleaved from the solid support by removing the group, and then isolated and purified. and annealing the sense or antisense strand of the nucleic acid.

[0378] In some embodiments, the P atom in formula A59 is a sense atom in the siRNA. The method for preparing the siRNA complex shown in formula (308) is as follows: (1) A compound of formula (321) (the compound of formula (321) is a compound having a protected hydroxyl group at R4) Si-OR k and a first functional group having a structure represented by formula (C1') or (C3'): The hydroxy-protecting group R k Remove the The deprotected product is converted to a nucleoside under coupling reaction conditions and in the presence of a coupling reagent. and contacting the monomer to obtain a nucleoside monomer that is bound to the solid support by a conjugate molecule. thing, (2) Starting with the nucleoside monomer that is bound to the solid support by the conjugated molecule, The sense strand of siRNA was synthesized by phosphoramidite solid-phase synthesis in the '-5' direction. To do, (3) Synthesize the antisense strand of siRNA by phosphoramidite solid-phase synthesis. To do so, (4) The sense and antisense strands of the siRNA are isolated and annealed to give the following formula (308): The method includes obtaining a siRNA complex as shown in

[0379] In step (1), the protecting group R in the compound of the above formula (321) k How to remove The method includes contacting a compound of formula (321) with a deprotecting reagent under deprotecting conditions. The conditions are that the temperature is 0 to 50° C., in some embodiments 15 to 35° C., and the reaction time is 30 In some embodiments, the deprotection reagent is trifluoromethylphenyl. One or more selected from the group consisting of dichloroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid. In some embodiments, dichloroacetic acid is used. The molar ratio of the compound is 10:1 to 1000:1, and in some embodiments, 50:1 to The ratio is 500:1.

[0380] The coupling reaction conditions and coupling reagents are suitable for the above coupling reaction. In some embodiments, solid phase synthesis is employed. The same conditions and reagents may be used as in the coupling reaction in the method.

[0381] In some embodiments, the coupling reaction conditions include a reaction temperature of 0 to 50° C. In some embodiments, the reaction temperature is 15 to 35° C. The molar ratio of the 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 is 1:1 to 1:50. In some embodiments, the ratio is 1:3 to 1:10, and the reaction time is 200 to 3000 seconds, and in some embodiments, 500 to 1500 seconds. 5-Ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole In some embodiments, one or more of the following are selected from the group consisting of 5-ethylthio 1H-tetrahydrofuran, The coupling reaction may be carried out in an organic solvent, is one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane In some embodiments, anhydrous acetonitrile is used. In contrast, the dosage of the organic solvent is 3 to 50 L / mol, and in some embodiments, 5 to 20 L. / mol.

[0382] In step (2), the nucleic acid prepared in the above step is synthesized by a phosphoramidite nucleic acid solid phase synthesis method. Starting with a nucleoside monomer that is bound to a solid support by a conjugated molecule, 3'-5' The sense strand SS of the second siRNA complex is synthesized in the direction of the nucleotide sequence of the complex. The 3′ end of the sense strand is linked to the 3′ end of the

[0383] Other conditions for the solid phase synthesis in steps (2) and (3) include deprotection of nucleoside monomers. Protection conditions, type and amount of deprotection reagent, coupling reaction conditions, type and amount of coupling reagent Dosage, capping reaction conditions, type and dosage of capping reagent, oxidation reaction conditions, oxidation reagent The types and dosages of drugs, sulfurization reaction conditions, and types and dosages of sulfurization reagents, which are commonly used in the field, A variety of reagents, doses and conditions are employed.

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

[0385] The deprotection conditions for the nucleoside monomers are a temperature of 0 to 50° C., and in some embodiments, 1 The temperature is 5 to 35° C., and the reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds. and the deprotection reagent is trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, chloroacetic acid and in some embodiments, dichloroacetic acid. The molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support is It may be from 2:1 to 100:1, and in some embodiments is from 3:1 to 50:1.

[0386] The coupling reaction conditions are at a temperature of 0 to 50° C., and in some embodiments, 15 to 35° C. The molar ratio of the nucleic acid sequence bound to the solid support to the nucleoside monomer is 1:1 to 1: 50, and in some embodiments 1:5 to 1:15, bound to a solid support. The molar ratio of the nucleic acid sequence to the coupling reagent is 1:1 to 1:100, in some embodiments The reaction time and the selection of coupling reagents are the same as above. do.

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

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

[0389] After all nucleoside monomers have been coupled and before annealing, the method further comprises: The method further comprises isolating the sense and antisense strands of A. Methods for isolating are known to those skilled in the art. Generally, the synthesized nucleotide sequence is cleaved from the solid support and the base is phosphorylated. This includes removing the protecting groups on the acid groups and on the ligands, purifying and desalting.

[0390] The synthesized nucleotide sequence is cleaved from the solid support and the bases, phosphate groups and ligands are removed. The above protecting groups can be removed by standard cleavage and deprotection methods used in siRNA synthesis. For example, the resulting solid support-bound nucleotide sequence may be dissolved in concentrated aqueous ammonia. In the deprotection process, the protecting group YCOO- of A46 to A54 is converted to a hydroxy group. and converting the S1 group to the corresponding M1 group to obtain the siRNA complex shown in formula (308). Here, the concentrated ammonia water is 25 to 30% by weight of ammonia water. The amount of concentrated ammonia water is 0.2 ml / mL for the siRNA sequence of interest. It may be from μmol to 0.8 ml / μmol.

[0391] If the synthesized nucleotide sequence has at least one 2'-TBDMS protection, The method comprises the steps of: cleaving the nucleotide sequence from the solid support by cleaving the nucleotide sequence with triethylamine trihydrofluoride; This further comprises removing the 2'-TBDMS protection by contacting the In this case, the corresponding nucleotide in the obtained siRNA sequence of interest contains two free The dose of pure triethylamine trihydrofluoride is The concentration may be 0.4 ml / μmol to 1.0 ml / μmol for the corresponding siRNA sequence. In this manner, 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, preparative ion chromatography. Purification of nucleic acids using a chromatographic purification column and gradient elution with NaBr or NaCl The product was collected, combined, and desalted using a reversed-phase chromatography purification column. It is possible.

[0393] In the thus obtained siRNA complex represented by formula (308), the nucleotide A non-bridging oxygen atom or sulfur atom in the phosphodiester or thiophosphodiester bond between The yellow atom is essentially bound to a sodium ion, and is represented by the formula (308): The NA complex exists primarily as a sodium salt. and replacing the sodium ions with hydrogen ions and / or other cations to form other types of formulas. The cation can be used in the same manner as described above. That is correct.

[0394] During 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, ion exchange chromatography Nucleic acid purity was detected by liquid chromatography tandem mass spectrometry (LCMS). The molecular weight can be measured by C-MS.

[0395] Annealing methods are also well known to those skilled in the art. The sense strand (S strand) and antisense strand (AS strand) were mixed in an equal molar ratio in water for injection and incubated for 70-95 min. C. and then cooled to room temperature, allowing hydrogen bonds to form a double-stranded structure. In this manner, the siRNA complex shown in formula (308) can be obtained.

[0396] After obtaining the siRNA complex, in some embodiments, the siRNA complex is subjected to, for example, liquid chromatography. The synthesized compound (30) was analyzed by molecular weight detection using methods such as HPLC-tandem mass spectrometry. 8) and characterized the siRNA complex. The siRNA complex shown in formula (308) was synthesized. Confirm that the sequence of the NA is the sequence of the desired siRNA, e.g., one of the sequences shown in Table 1. It is also possible.

[0397] The compound of formula (321) can be prepared by the reaction of the esterification reaction with a base in an organic solvent. and contacting the compound represented by formula (313) with a cyclic acid anhydride in the presence of an esterification catalyst. followed by ion exchange and isolation to obtain the compound of formula (321). can be obtained by:

[0398] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1、 The definition and selectable range of each S1 are as described above. R6 is a group that provides R4 in formula (321). In some embodiments, , R6 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 Combined with O to form one is any group to which a free hydroxy group of k is a hydroxy protecting group. In this case, R4 includes a first functional group as a hydroxy protecting group and a second functional group, A compound of formula (321) in which the second functional group contains a structure represented by formula (C1) or (C2) is obtained. can be done.

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

[0401] In some embodiments, the organic solvent is an epoxy solvent, an ether solvent, a haloperidol solvent, or a cyclohexane solvent. Alkyl halogenated solvents, dimethyl sulfoxide, N,N-dimethylformamide and N , N-diisopropylethylamine. In some embodiments, The epoxy solvent is dioxane and / or tetrahydrofuran, and the acetone is The tert-butyl ether solvent is ethyl ether and / or methyl tert-butyl ether, The halogenated alkyl solvents are dichloromethane, trichloromethane and 1,2-dichloromethane. In some embodiments, the organic solvent is one or more of dichloromethane. The amount of the organic solvent is 3 to 3% by weight based on the compound represented by the formula (313). ~50 L / mol, and in some embodiments, 5-20 L / mol.

[0402] In some embodiments, the cyclic acid anhydride is succinic anhydride, glutaric anhydride. anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments , succinic anhydride. The compound represented by the formula (313) and the cyclic acid anhydride The ratio of the fluorine atom to the fluorine atom 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, e.g. For example, the catalyst may be 4-dimethylaminopyridine. ) is 1:1 to 10:1 in some embodiments. , 2:1~5:1.

[0404] In some embodiments, the base is any inorganic base, organic base, or combination thereof. Considering the solubility and stability of the product, the base may be, for example, In some embodiments, the tertiary amine is a triethylamine. The tertiary amine and the formula (3 13) is 1:1 to 20:1 in molar ratio, and in some embodiments, The ratio is between 3:1 and 10:1.

[0405] The ion exchange reaction is carried out by reacting the compound of formula (321) with the desired carboxylic acid or carboxylate. The ion exchange method is known to those skilled in the art, and a suitable ion exchange Using the solution and exchange conditions, M + It is possible to obtain a complex molecule having a cation, which is described in detail here. In some embodiments, the ion exchange reaction is carried out using triethylamine. The concentration of the triethylamine phosphate solution is 0.2 to 100%. 0.8M, and in some embodiments, the triethylamine phosphate solution The concentration is 0.4 to 0.6 M, and the triethylamine is The dosage of the phosphate solution is 3-6 L / mol, and in a further embodiment 4-5 L / mol. She is an office lady.

[0406] Isolating the compound of formula (321) from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the mixture is subjected to a chromatographic method. The compound of formula (321) can be isolated by, for example, (1) normal phase purification silica gel 200-300 mesh silica gel packing material, 1 wt% triethylamine Gradient elution with chloromethane:methanol = 100:18 to 100:20, or 2) Reverse phase purification: C18, C8 reverse phase packing was used in methanol:acetonitrile = 0.1:1 ~ The compounds were isolated using two chromatographic conditions: 1:0.1 gradient elution and 2:1 gradient elution. In some embodiments, the solvent can be directly removed to provide a crude product of compound of Formula (321). The crude product can be used as is in the subsequent reaction.

[0407] In some embodiments, the method for preparing a compound of Formula (321) comprises the steps of: In an organic solvent, the above ion exchange reaction is carried out in the presence of a condensation agent, a condensation catalyst and a tertiary amine. The product obtained by the reaction is then contacted with a solid phase carrier containing an amino group or a hydroxy group. In this case, R4 includes a first functional group and a second functional group, and the first The functional group comprises a hydroxy protecting group and the second functional group comprises the structure shown in formula (C1'): Compound (321) is obtained.

[0408] The solid phase support is one of the supports used in solid phase synthesis of siRNA. Some are known to those of skill in the art. For example, the solid phase support may have an active hydroxy or amino group. In some embodiments, the solid support may be selected from a solid support that includes a functional group. In some embodiments, the amino resin is a hydroxy resin. The resin or hydroxy resin has a particle size of 100 to 400 mesh, and the surface The amino or hydroxyl group loading parameter is 0.2 to 0.5 mmol / g. The dosage ratio of the compound represented by the formula (321) to the solid phase carrier is 10 to 400 μmol. Compound / gram of solid support (μmol / g). The dosage ratio of the compound represented by formula (321) to the solid support 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 acetonitrile, an epoxy solvent, an ether, Solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide In some embodiments, the amine is one or more of N,N-diisopropylethylamine and N,N-diisopropylethylamine. In the embodiment, 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 dichloromethane, trichloromethane, and 1,2 In some embodiments, the organic solvent is one or more of: is acetonitrile. The amount of the organic solvent is 20 for the compound of formula (321). In some embodiments, the concentration is 50 to 100 L / mol. do.

[0410] In some embodiments, the condensing agent is benzotriazol-1-yl-oxy Tripyrrolidino-phosphonium hexafluorophosphate / ester (benzotri iazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate, PyBop), 3-diethoxyphosphoryl -1,2,3-Benzoxazol-4(3H)-one (3-(Diethoxyphos phoryloxy)-1,2,3-benzotriazin-4(3H)-one, DEPBT) and / or O-benzotriazole-tetramethyluronium hexafluoro O-benzotriazol-1-yl-tetramethylbenzoate / ester thyluronium hexafluorophosphate), In some embodiments, the condensing agent is O-benzotriazole-tetramethyl ether. The condensation agent is tetrafluoromethane hexafluorophosphate / ester. The molar ratio of the compound to be treated is 1:1 to 20:1, and in another embodiment, 1:1 to 5: The answer is 1.

[0411] In some embodiments, the tertiary amine is triethylamine and / or N, N-diisopropylethylamine, and in some embodiments, N,N-diisopropyl propylethylamine, and the molar ratio of the tertiary amine to the compound represented by formula (321) The ratio of the fluorine-containing polymer to the fluorine-containing polymer is from 1:1 to 20:1, and in some embodiments, from 1:1 to 5:1.

[0412] In some embodiments, the method for preparing a compound of Formula (321) comprises a capping reaction The resulting condensation product is reacted with a capping reagent and an acylation catalyst in an organic solvent under the conditions described above. and isolating to obtain a compound of formula (321). The function of the capping reaction is to avoid the generation of unwanted by-products in subsequent reactions. The first step is to remove any active reactive functional groups that have not yet fully reacted in order to obtain a The conditions for the capping reaction are a reaction temperature of 0 to 50° C., and in some embodiments, 15 to 3 5° C., and the reaction time is 1 to 10 hours, and in some embodiments, 3 to 6 hours. The capping reagent is a capping reagent known to those skilled in the art and used in siRNA solid-phase synthesis. This is also fine.

[0413] In some embodiments, the capping reagent is capping reagent 1 (cap 1) and capping reagent 2 (cap2), where capping reagent 1 is N-methyl imidazole, and in some embodiments, pyridine of N-methylimidazole. It is supplied as a pyridine / acetonitrile mixture with a volume ratio of 1:1. 10 to 1:1, and in some embodiments, 1:3 to 1:1, and pyridine and The volume ratio of the total volume of acetonitrile to N-methylimidazole is 1:1 to 10:1. In some embodiments, the ratio is 3:1 to 7:1. In some embodiments, the capping reagent 2 is acetic anhydride. It is supplied as a solution in acetonitrile with a volume ratio of acetic anhydride to acetonitrile of 1:1. to 1:10, and in a further embodiment from 1:2 to 1:6.

[0414] In some embodiments, the N-methylimidazole is The ratio of the volume of the mixed solution to the mass of the compound of formula (321) is 5 ml / g to 50 ml / g. In some embodiments, the acetic anhydride acetone concentration is 15 ml / g to 30 ml / g. The ratio of the volume of the trinitrile solution 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, the capping reagent is an equimolar mixture of acetic anhydride and N In some embodiments, the organic solvent is acetonitrile. Nitriles, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfur 1) 1-Hexoxide, N,N-dimethylformamide and N,N-diisopropylethylamine In some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent is 10 to 50 L / mol, or In some embodiments, it is 5 to 30 L / mol.

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

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

[0418] In some embodiments, the method for preparing the conjugated molecule of formula (321) comprises the steps of: In the present invention, under coupling reaction conditions and in the presence of a coupling reagent, The compound shown is contacted with a phosphorodiamidite and isolated to give the compound shown in formula (321). In this case, R4 includes a first functional group and a second functional group, and the first functional group is wherein the functional group comprises a hydroxy protecting group and the second functional group comprises a structure shown in formula (C3): Compound (321) is obtained.

[0419] In some embodiments, the coupling reaction conditions may be at a temperature of 0 to 50° C. The reaction temperature is usually, for example, 15 to 35° C. The reaction temperature is preferably 15 to 35° C. The ratio of the amines may be 1:1 to 1:50, for example, 1:5 to 1:15, and is represented by the formula (313): The molar ratio of the compound to the coupling reagent may be 1:1 to 1:100, for example 1 The reaction time may be 200 to 3000 seconds, for example, 500 The phosphorodiamidite is, for example, bis(diisopropylamino )(2-cyanoethoxy)phosphine may be used, which may be purchased commercially, or It may be synthesized by methods known in the art. The coupling reagent is 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole The coupling agent is one or more of the above-mentioned compounds, for example, 5-ethylthio-1H-tetrazole. The pulling reaction may be carried out in an organic solvent, and the organic solvent may be anhydrous acetonitrile, anhydrous One or more selected from water, DMF, and anhydrous dichloromethane, for example, anhydrous acetonitrile. In some embodiments, the compound of formula (313) is The amount of the organic solvent is 3 to 50 L / mol, and may be, for example, 5 to 20 L / mol. By carrying out the coupling reaction, the hydroxy group in the compound of formula (313) is With a phosphoramidite to form a phosphoramidite group. In this embodiment, the solvent can be directly removed to obtain the crude compound of formula (321), The crude product can be used as is in the subsequent reaction.

[0420] In some embodiments, the method for preparing a compound of Formula (321) comprises a coupling reaction The product obtained by isolating the product obtained by reacting the product with the product obtained by reacting ... and then contacting the solid phase carrier with a hydroxy group-containing solid phase carrier. The compound of formula (321) is obtained by carrying out a cycloaddition reaction, an oxidation reaction, and isolating the compound. and a second functional group, the first functional group comprising a hydroxy protecting group and the second functional group comprising a A compound of formula (321) in which the functional group has the structure shown in formula (C3') is obtained.

[0421] In some embodiments, the solid support is a suitable substrate for solid phase synthesis of nucleic acids as known in the art. For example, a commercially available general-purpose solid phase support (NittoPhase) that has been deprotected is ase(R)HL UnyLinker TM 300 Oligonucleotides ide Synthesis Support, Kinovate Life Science Nces Corporation, the structure of which is shown in formula B80.

[0422] [ka]

[0423] Deprotection reactions are known to those skilled in the art. In some embodiments, the deprotection conditions are The temperature is 0 to 50°C, for example 15 to 35°C, and the reaction time is 30 to 300 seconds, for example 50 to The deprotection reagents were trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid. and in some embodiments, one or more selected from: The deprotection reagent is dichloroacetic acid. -dimethoxytrityl) protecting group is 2:1 to 100:1 in molar ratio, for example, 3:1 to The ratio of the deprotection ratio to the ratio of the reaction mixture to the reaction mixture is 50:1. By carrying out the deprotection, reactive A free hydroxy group is obtained, making it accessible for the next coupling reaction.

[0424] The coupling reaction conditions and the selection of coupling reagents are as described above. By carrying out a coupling reaction, the free hydroxyl group formed in the deprotection reaction and the phosphoryl group The midite groups are reacted to form phosphite linkages.

[0425] In some embodiments, the capping reaction conditions include a temperature of 0 to 50° C., e.g., 1 The temperature is 5 to 35° C., the reaction time is 5 to 500 seconds, for example, 10 to 100 seconds, The capping reaction is carried out in the presence of a capping reagent. The choice and dosage of the capping reagent are as described above. As follows.

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

[0427] In some embodiments, R6 is one of the groups of formula B7 or B8.

[0428] [ka] In the formula, q2 is defined as above.

[0429] In this case, the compound represented by formula (313) can be reacted in an organic solvent under amidation reaction conditions: and amidation reaction: In the presence of a condensing agent and a tertiary amine, a compound represented by formula (314) is converted into contacting the compound represented by formula (A-1) or the compound represented by formula (A-2) with the compound represented by formula (A-3) and then isolating the compound represented by formula (A-4); It can be obtained by the preparation method described above.

[0430] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L1, S1, q2 and R k The definitions and available ranges of each are as described above. be.

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

[0432] In some embodiments, the organic solvent is an alcohol solvent, an epoxy solvent, Ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethyl formamide and N,N-diisopropylethylamine. In some embodiments, the alcohol solvent is methanol, ethanol, propanol, or the like. In some embodiments, the epoxy is one or more of ethanol. The alkoxy solvent, in some embodiments, is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or is methyl tert-butyl ether. The halogenated alkyl solvents include In the embodiment, 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); In a further embodiment, it is from 3 to 20 L / mol.

[0433] In some embodiments, the amidation reaction condensing agent is benzotriazole-1- Il-oxytripyrrolidino-phosphonium hexafluorophosphate / ester, 3 -Diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one, 4-(4 ,6-Dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (4-(4,6 -dimethoxytriazin-2-yl)-4-methylmorpholi ne hydrochloride), 2-ethoxy-1-ethoxycarbonyl-1,2 -Dihydroquinoline (EEDQ) or O-benzotriazole-tetramethyluronium In a further embodiment, 3-diethoxy hexafluorophosphate / ester. The amidation is diphosphoryl-1,2,3-benzoxazol-4(3H)-one. The molar ratio of the reaction condensation agent to the compound represented by formula (314) may be 1:1 to 10:1. In some embodiments, the ratio is from 2.5:1 to 5:1.

[0434] In some embodiments, the tertiary amine is triethylamine or N,N-diethylamine. In a further embodiment, N,N-diisopropyl ethyl amine. The molar ratio of the tertiary amine to the compound represented by formula (314) is 3: It is 1 to 20:1, and in some embodiments, it is 5:1 to 10:1.

[0435] In some embodiments, the compounds of formula (A-1) and formula (A-2) may be any suitable For example, R k When is a DMTr group, glyceric acid The compound of formula (A-1) can be prepared by reacting calcium with DMTrCl. Similarly, 3-amino-1,2-propanediol was contacted with a cyclic acid anhydride, and then DMTr was added. Cl to prepare a compound of formula (A-2), The acid anhydride may be a cyclic acid anhydride having 4 to 13 carbon atoms, and in some embodiments, 4 to 8 carbon atoms. As can be easily understood by those skilled in the art, the selection of the cyclic acid anhydride is based on the compound (A-2). For example, the cyclic anhydride may be succinic anhydride. When the cyclic acid anhydride is glutaric anhydride, q2=1. 2, which may be inferred.

[0436] In some variations, the compound of formula (314) can be prepared by reacting the cyclic anhydride, 3- By reacting sequentially with amino-1,2-propanediol and DMTrCl, 313) can also be prepared. These modifications do not affect the structure and function of the compound of formula (313) and are within the scope of the present invention. This can be easily achieved by the vendor using the above method.

[0437] As above, the compound of formula (313) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (313) can be isolated by a chromatography method, for example, (1) normal phase purification. Silica gel: 200-300 mesh silica gel packing material, petroleum ether: ethyl acetate Alcohol:dichloromethane:N,N-dimethylformamide=1:1:1:0.5~1:1:1 (2) Reverse-phase purification: C18 and C8 reverse-phase packings were used with a gradient elution of 0.6. The two chromatographs were eluted with a gradient of ethanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly isolated using chromatographic conditions. The crude product of the compound of formula (313) can be obtained by removing the crude product, which can be directly used for the subsequent It can be used in the reaction.

[0438] In some embodiments, the compound represented by Formula (314) is reacted in an organic solvent with Amidation reaction In the presence of a condensing agent and a tertiary amine, under condensation reaction conditions, a compound represented by formula (320) is obtained. contacting a compound represented by formula (316) with a compound represented by formula (316), followed by isolation. It can be obtained by law.

[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) can be prepared, for example, by the method described in J. Am. Chem. Soc. 2014, 136, 16958-16961 may be used, or a compound represented by the formula (31 6) The compounds can be prepared by a person skilled in the art by various methods, for example, those described in U.S. Pat. With reference to the method disclosed in Example 1 of No. 8,106,022 B2, several formulas (316 The compounds of the present invention can be prepared by the methods described above, the entire contents of which are incorporated herein by reference. is incorporated into the book.

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

[0442] Considering the structure of the desired product, compound of formula (314), the compound shown in formula (316) above The molar ratio of the compound to be reacted with the compound represented by the formula (320) is It should be determined based on the sum of n1 and n3. In some embodiments, for example, , when n1 + n3 = 3, to ensure that the reaction is complete and not excessive, 316) and the compound represented by the formula (320) in a molar ratio of 3:1 to 3 0.5:1, and in some embodiments, 3.01:1 to 3.15:1. be.

[0443] In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, an ethylene oxide solvent, Ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethyl ether and N,N-diisopropylethylamine, The alkoxy solvent, in some embodiments, is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or is methyl tert-butyl ether, and the halogenated alkyl solvent is In the embodiment, dichloromethane, trichloromethane and 1,2-dichloroethane In some embodiments, the organic solvent is dichloromethane. For the compound of formula (320), the amount of the organic solvent is 3 to 50 L / mol. In some embodiments, the concentration is 5 to 20 L / mol.

[0444] In some embodiments, the amidation reaction condensing agent is benzotriazole-1- Il-oxytripyrrolidino-phosphonium hexafluorophosphate / ester, 3 -Diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPB T), O-benzotriazole-tetramethyluronium hexafluorophosphate / Ester, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine salt or 1-hydroxybenzotriazole, in further embodiments. In the present invention, benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluoride It is a mixture of fluorophosphates / esters and 1-hydroxybenzotriazole. Triazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphonium The phosphate / 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) is 1:1 to 3:1. In some embodiments, the ratio is 1.05:1 to 1.5:1.

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

[0446] Similarly as above, 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 chromatograph may be The compound of formula (314) can be isolated by a chromatography method, for example: (1) normal phase purification Silica gel: 200-300 mesh silica gel packing material, dichloromethane:methanol (2) Reverse phase purification: C18, C 8. Elute the reversed-phase packing with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the chromatographic conditions are: In the above, the solvent can be directly removed to obtain the crude product of the compound of formula (314), The product can be used as is in subsequent reactions.

[0447] Compounds of formula (320) can be obtained commercially or by methods known to those skilled 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 Inc.

[0448] The siRNA complexes of the present disclosure may be used in combination with other pharma- ceutically acceptable additives. The additive may be one or more of a variety of agents 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 siRNA and / or drug compositions of the present disclosure. and / or the siRNA complex, a disease or physiological condition caused by a uric acid metabolic disorder In some embodiments, the present invention provides a method for the preparation of a medicament for the treatment and / or prevention of In the above, the disease or physiological condition caused by abnormal uric acid metabolism is hyperuricemia or gout.

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

[0451] siRNA active components of the present disclosure can be administered to a subject in need thereof to induce RNase activity. Prevents and / or treats uric acid metabolism disorders or diseases or physiological conditions caused by uric acid metabolism disorders through the A interference mechanism. Therefore, the siRNA and / or The drug composition and / or the siRNA complex is / are intended to treat or prevent uric acid metabolism disorders or diseases caused by uric acid metabolism disorders. is used in the prevention and / or treatment of physiological conditions, or disorders of uric acid metabolism or conditions resulting from disorders of uric acid metabolism They can be used in the preparation of medicaments for the prevention and / or treatment of diseases or physiological conditions. In some embodiments, the uric acid metabolism disorder or the disease or physiological condition caused by the uric acid metabolism disorder is , hyperuricemia or gout.

[0452] As used herein, the term "pharmaceutical administration / administration" refers to the administration of siRNA, drug compositions of the present disclosure. and / or localizing at least a portion of the siRNA complex to a desired site to produce a desired effect. The siRNA, drug composition and / or siRNA complex of the present disclosure can be administered by any method or route that allows the siRNA to be administered. Suitable routes of administration for the methods of the present disclosure include topical administration and Local administration generally results in more siRNA being delivered to the subject than to the systemic circulation of the subject. The complex is delivered to a specific site, but systemic administration of the siRNA, drug composition of the present disclosure and / or the siRNA complex is delivered to the basal systemic circulation of the subject. To provide a means for preventing and / or treating diseases or physiological conditions caused by abnormalities or abnormalities in uric acid metabolism. In view of the above, in some embodiments, the drug is delivered to the liver. Adopt an administration method that can achieve this.

[0453] Administration to a subject can be by any suitable route known in the art, including, but not limited to, Oral or non-gastrointestinal routes, e.g., intravenous, intramuscular, subcutaneous, transdermal, intratracheal Intravenous (aerosol), pulmonary, nasal, rectal and local (buccal and sublingual) The frequency of administration may be daily, weekly, or biweekly. , once or more per three weeks, once a month or once a year.

[0454] The dosage of the siRNA, drug composition or siRNA complex described in this disclosure may be determined by methods known in the art. The dose may be a usual dose for the patient, said dose being determined according to various parameters, in particular the age of the subject. Toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Measure sex and treatment effects, e.g., LD 50 (the dose that kills 50% of the colony) and ED5 0 (refers to the dose that can induce 50% of the maximum reaction intensity in a quantitative response, In qualitative reactions, this refers to the dose at which a positive reaction occurs in 50% of experimental subjects. Based on data from cell culture assays and animal studies, a range of human dosages is available. The range can be obtained.

[0455] Administering the siRNA, drug composition, and / or siRNA complex described in this disclosure For example, male or female, 6 to 12 weeks old, weighing 18 to 25 g, C57BL / 6J or For ob / ob mice weighing 30–45 g, the following amounts of siRNA were administered: (i) siRNA complex; For the combination, the siRNA dosage may be 0.001 to 100 mg / kg body weight. In some embodiments, the dose is 0.01 to 50 mg / kg body weight, and in some embodiments, the dose is 0.01 to 50 mg / kg body weight. In some embodiments, the dose is 0.05 to 20 mg / kg body weight, and in some embodiments, the dose is 0.1 to 20 mg / kg body weight. 15 mg / kg body weight, and in some embodiments, 0.1 to 10 mg / kg g body weight; and (ii) a pharmaceutical composition formed by the siRNA and a pharma- ceutical acceptable carrier. For the composition, the siRNA dosage may be 0.001 to 50 mg / kg body weight; In some embodiments, the dose is 0.01 to 10 mg / kg body weight, and in some embodiments, the dose is 0.01 to 10 mg / kg body weight. 0.05 to 5 mg / kg body weight, and in some embodiments 0.1 to 3 mg / kg body weight It is.

[0456] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or contacting a cell with an effective amount of the siRNA complex of the present disclosure. The drug composition and / or the siRNA complex are introduced into the cells, and the cells are amplified by the RNA interference mechanism. The present invention relates to a method for inhibiting the expression of XO gene in a cell. The present invention provides a method for inhibiting the expression of the O gene. The cells may be selected from cancer cell lines such as Huh7 or isolated primary hepatocytes. In embodiments, the cells are CAL-27 cells.

[0457] The method provided by the present disclosure suppresses expression of the XO gene in a cell. The siRNA dosage in the modified siRNA, drug composition and / or siRNA complex is Generally, it can reduce the expression of the target gene, and the target cell surface can be treated with a concentration of 1 pM to 1 μM, or 0. 0.1 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to about 5 nM The amount required to achieve that local concentration will depend on the delivery method, delivery site, and , the number of cell layers between the delivery site and the target cell or tissue, the route of delivery (local or systemic), etc. The concentration at the delivery site varies depending on a variety of factors. The concentration may be significantly higher than that of the

[0458] <Kit> The present disclosure relates to at least one of the modified siRNAs, drug compositions, and siRNA complexes of the present disclosure. A kit is provided that contains an effective amount of the species.

[0459] In some embodiments, the kits described herein include a container containing the modified siRNA. In some embodiments, the kits described herein can include In some embodiments, the container may include a container providing a pharma- ceutically acceptable excipient. Thus, the kit may contain other ingredients, such as stabilizers or preservatives. In some embodiments, the kits described herein include the modified s At least one other therapeutic agent may be included in a container separate from the container providing the iRNA. In some embodiments, the kit comprises a modified siRNA and a pharma- ceutically acceptable carrier and and / or instructions for mixing the composition and / or additives or other ingredients, if present.

[0460] In the kit of the present disclosure, the modified siRNA and a pharma- ceutically acceptable carrier and / or Additives, and the modified siRNA, drug composition and / or siRNA complex, and / or The pharma- ceutically acceptable excipient may be in any form, for example, liquid form, dried form, or lyophilized form. In some embodiments, the modified siRNA and A pharma- ceutically acceptable carrier and / or excipient, and the pharmaceutical composition and / or siR The NA complex and any pharma- ceutically acceptable excipients are essentially clean and / or sterile. In some embodiments, sterile water can be provided in the kits 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. do not have. EXAMPLES

[0462] Unless otherwise specified, all reagents and media used in the following examples are commercially available products. The nucleic acid electrophoresis and real-time PCR procedures used are all performed by Molecular cular Cloning(Cold Spring Harbor Laborat This is carried out with reference to the method described in (Ory Press, 1989).

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

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

[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, the siRNA complex L10-siXOi1M1S was synthesized. The NA complex was formed after the L-9 complex molecule was complexed with siRNA numbered siXOi1M1S. The siRNA complex formed by the siRNA synthesis is shown in FIG. 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-2 100.0 g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8 mmol) 1000 ml 100 ml of anhydrous pyridine, and 540 ml of acetic anhydride (purchased from Enox, The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was poured into 10 L of ice water. The cake was washed with 2 L of ice water and then cooled to room temperature until completely dissolved. Add acetonitrile / toluene mixed solvent (acetonitrile:toluene volume ratio = 1:1) The solvent was evaporated to dryness to obtain 130.0 g of a white solid product, GAL-2.

[0471] (1-1-1b) Synthesis of GAL-3 The GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was Dissolve in 3 ml of anhydrous 1,2-dichloroethane and immerse in an ice-water bath under nitrogen protection. 0 g of TMSOTf (CAS number: 27607-77-8, purchased from Macklin, 10 8.0 mmol) was added and the reaction was carried out at room temperature overnight.

[0472] The reaction mixture was diluted with 400 ml of dichloromethane, filtered through diatomaceous earth, and then added to 1 L of saturated charcoal. Add an aqueous solution of sodium hydrogen carbonate, stir evenly, separate the organic phase, and dichloroethane into the aqueous phase. Extract twice with 300 ml each time, combine the organic phases, and add 300 ml of saturated The mixture was washed with an aqueous solution of sodium bicarbonate and 300 ml of saturated saline, and the organic phase was separated and dried. The mixture was dried over sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to give 26.9 g of a pale yellow, viscous starch syrup-like product. The product GAL-3 was obtained.

[0473] (1-1-1c) Synthesis of GAL-4 The GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was Dissolve in 6 ml of anhydrous 1,2-dichloroethane and add 30 g of dried 4 Å molecular sieve powder. 9.0 g of 5-hexen-1-ol (CAS number: 821-41-0, Adamas- Beta Co., 89.9 mmol) was added, stirred at room temperature for 30 minutes, and then cooled in an ice bath and nitrogen. 9.08 g of TMSOTf (40.9 mmol) was added under protection and reacted at room temperature overnight with stirring. The 4 Å molecular sieve powder was removed by filtration, and the filtrate was diluted with 300 ml of dichloromethane. Filter through diatomaceous earth, add 500 ml of saturated aqueous sodium bicarbonate solution, stir for 10 minutes, and wash. The organic phase was separated, the aqueous phase was extracted once with 300 ml of dichloroethane, and the organic phase was evaporated. Then, 300 ml of saturated sodium bicarbonate solution and 300 ml of saturated saline solution were used. The organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure. The yellow syrup-like product GAL-4 was obtained in an amount of 0.3 g, and was used in the next oxidation reaction without purification. I did.

[0474] (1-1-1d) Synthesis of GAL-5 GAL-4 (14.9 g, 34 0.7 mmol) was dissolved in a mixture of 77 ml of dichloromethane and 77 ml of acetonitrile. The mixture was dissolved in 103 ml of deionized water and 29.7 g of sodium periodate (CA S number: 7790-28-5, purchased from Aladdin, 138.8 mmol) was added. The mixture was stirred in an ice-water bath for 10 minutes, and ruthenium(III) chloride (CAS number: 14898-6 7-0, purchased from Energy Co., 238 mg, 1.145 mmol) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with 300 ml of water and stirred, and saturated sodium bicarbonate was added. The pH was then adjusted to about 7.5, the organic phase was separated and discarded, and the aqueous phase was washed once with dichloromethane. The organic phase was discarded. The pH of the aqueous phase was adjusted to approximately 3 with solid citric acid. The mixture was adjusted to 0.5% and extracted three times with dichloromethane, each time with 200 ml of dichloromethane. The organic phases were combined and The mixture was dried over sodium sulfate and the solvent was evaporated to dryness under reduced pressure to give 6.85 g of a white foamy solid product G. Got AL-5. 1H NMR (400 MHz,DMSO) δ 12.01 (b r,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,3 H), 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 process ( GAL-5 (72.819 g, 162.75 mmol, multiple loci) obtained in 1-1-1 Dissolve the above mixture (a combination of the products from the previous batch) in 525 ml of dichloromethane and add diisopropyl Ethylamine (DIEA, 44.782g, 346.50mmol), Benzotriazol 1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate / ess PyBOP (90.158 g, 173.25 mmol) and hydroxybenzotriester Add azole (HOBt, 23.410 g, 173.25 mmol) and react at room temperature for 4 hours. Then, 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, and the organic phases were combined and washed with anhydrous sulfuric acid. The crude product was obtained by drying over sodium hydroxide, filtering, and then evaporating the solvent under reduced pressure to dryness. 200-300 mesh normal phase silica gel was used, and silica was purified with 10 wt% triethylamine. Neutralize the acidity of the gel, equilibrate the column with 1 wt‰ triethylamine, and then add dichloromethane. Elute with a gradient of 100:25 to 100:40 methanol, and collect the product eluate. The extract was evaporated to dryness under reduced pressure to give 38.8 g of pure L-8. 1 H NMR (400 MHz ,DMSO) δ 7.84 (d,J = 9.0 Hz,3H),7.27-7.2 3 (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),2.15-2.0 8 (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 N7O 30 , [M+H] + , Theoretical value: 1477.59, Actual value: Measurement: 1477.23.

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

[0478] DMTrCl (4,4'-bismethoxytrityl chloride, 101.65 g, 300 m mol) was dissolved in 1000 ml of anhydrous pyridine and DL-calcium glycerate hydrate was obtained. The reaction mixture was added to the flask (28.63 g, 100 mmol) and reacted at 45° C. for 20 hours. The reaction mixture was then filtered. The cake was rinsed with 200 ml of DCM, the filtrate was concentrated under reduced pressure to dryness, and the remainder was 1 of dichloromethane, and 0.5 M triethylamine phosphate (pH = 7.5-1.0). 8), wash twice with 200 ml each time, and then wash the aqueous phase with dichloromethane, 20 The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed. The mixture was evaporated to dryness under reduced pressure, and purified using a 200-300 mesh normal phase silica gel column. Alcohol:ethyl acetate:dichloromethane:methanol=1:1:1:0.35 to 1:1:1:0 The product eluate was collected and the solvent was evaporated to dryness under reduced pressure. Redissolve in dichloromethane and wash once with 200 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted once with 200 ml of dichloromethane, and the organic phases were combined and washed with anhydrous sodium sulfate. The mixture was dried over sodium, filtered, and the solvent was evaporated to dryness under reduced pressure, then the mixture was cooled to 50°C overnight using a vacuum oil pump. 7 g of a white solid product A-1 was obtained. 1 H 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,6 H),1.03 (t,J = 6.3 Hz,9H).MS m / z:C 24 H 23 O6, [MH] - , theoretical value: 407.15, measured value: 406.92.

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

[0480] L-8 (40 g, 27.09 mmol, multiple lots) obtained in step (1-1-2) The products were combined and A-1 (41.418 g, 81 0.27mmol) and dissolved in 271ml of dichloromethane, and Phosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (24. Add 318 g, 81.37 mmol of diisopropylethylamine (21.0 07g, 162.54mmol) was added, and the mixture was stirred at 25°C for 1.5 hours. Wash the organic phase with saturated sodium bicarbonate and the aqueous phase with dichloromethane, 50 ml at a time. The organic phase was washed with 150 ml of saturated saline, and the aqueous phase was extracted with 50 ml of dichloromethane. Extract once with methane, combine the organic phases, dry over anhydrous sodium sulfate, filter, and dissolve. The agent was evaporated to dryness under reduced pressure and then foam-dried overnight using a vacuum oil pump to obtain a crude product. 2 kg of 200-300 mesh normal phase silica gel was used, and 200 ml of triethylamine was added. The acidity of the silica gel was neutralized with amine, and the silica gel was then colored with petroleum ether containing 1 wt% triethylamine. The mixture was equilibrated with petroleum ether:ethyl acetate:dichloromethane:N,N-dimethylformamide. Gradient elution with mido = 1:1:1:0.5 to 1:1:1:0.6 was performed, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure to give 40.4 g of pure L-7. 1 H NMR (400 M Hz,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 N7O 35 , [M-DMTr] + ,Theoretical value: 1564.65,Actual value 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), amber Acid anhydride (4.288 g, 42.8494 mmol) and 4-dimethylaminopyridine ( DMAP, 5.235 g, 42.8494 mmol) was mixed in 215 mL of dichloromethane. Dissolve diisopropylethylamine (DIEA, 13.845 g, 10 Add 7.1235 mmol) and stir at 25°C for 24 hours. Add 800 ml of 0.5 M triethyl ether. The reaction mixture was washed with diethylamine 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 the crude product. Normal phase silica gel of 0 to 300 mesh was used, and the silica gel was dissolved in 1 wt% triethylamine. The acid was neutralized, the column was equilibrated with dichloromethane, and a dichloromethane solution containing 1 wt% triethylamine was added. Gradient elution was performed with chloromethane:methanol = 100:18 to 100:20, and the product eluate was After recovery, the solvent was evaporated to dryness under reduced pressure to obtain 31.0 g of pure L-9 complex molecule. 1 H.N.M. R (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),2.15-1.9 5 (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 N7 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 step, the L-9 conjugate molecule is bound to a solid support to form the L-10 compound. was prepared.

[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 (DI EA, 2.843 g, 22 mmol) was mixed and dissolved in 900 ml of acetonitrile. The reaction mixture was stirred at room temperature for 5 minutes, and aminomethyl resin (88 g, 100-200 mesh, Amino group loading amount 400 μmol / g, purchased from Nankai Waseisha Co., Ltd.) was added and incubated on a shaker at 25°C. The reaction was carried out at a rotation speed of 150 rpm for 18 hours, after which the cake was filtered and separated into 100 ml of DC Rinse twice with 300 ml of M, then rinse twice with 300 ml of acetonitrile. The mixture was rinsed three times with water and dried for 18 hours using a vacuum oil pump. Supplements (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) ) was added and the capping reaction was carried out. The mixture was placed in a shaker at 25°C and rotated at 150 rpm. The reaction was carried out at 0.05 °C for 5 h at 100 °C for 1 min, and the reaction mixture was filtered. The cake was diluted with acetonitrile at 300 mL / min. Rinse 3 times with 10 ml of water, evaporate the solvent under reduced pressure until dry, and then dry overnight under reduced pressure with a vacuum oil pump. 102 g, with a loading of 90.8 μmol / g of L-10 compound (i.e., bound to a solid support). L-9 complex molecule) was obtained.

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

[0487] (1-2) Synthesis of the sense strand of siRNA complex L10-siXOi1M1S Starting from the L-10 compound prepared in the above step, the solid-phase phosphoramidite method was used to The nucleotides are rotated one by one in the 3'-5' direction according to the nucleotide sequence of the sense strand. Each time a nucleoside monomer is bonded, deprotection, coupling Four reactions were carried out: capping, oxidation, or sulfurization. When the nucleoside monomer is bonded by ester, deprotection, coupling, etc. are performed when the next nucleoside monomer is bonded. Four reactions were performed: plucking, capping, and oxidation. When the nucleoside monomer is linked by an acid ester, it is protected, substituted, or substituted when linking the next nucleoside monomer. Four reactions were carried out: coupling, capping, and sulfurization. The synthesis conditions were as follows: did.

[0488] Nucleoside monomers were provided in acetonitrile at 0.1 M concentration for each deprotection reaction. The conditions were the same, that is, the temperature was 25°C and the reaction time was 70 seconds. A solution of dichloroacetic acid in dichloromethane (3% v / v) was used. , 4'-dimethoxytrityl protecting group is 5:1 molar ratio.

[0489] The coupling reaction conditions were the same for all reactions, with the reaction temperature at 25°C and the reaction mixture bound to the solid support. The molar ratio of the nucleic acid sequence to the nucleoside monomer is 1:10, and the nucleic acid sequence bound to the solid support is The molar ratio of the column to the coupling reagent was 1:65, and the reaction time was 600 seconds. The reagent was 5-ethylthio-1H-tetrazole (5-(Ethylthio)-1H-t etrazole (ETT) 0.5M solution in acetonitrile.

[0490] The capping conditions were the same for all of them, with the temperature at 25°C and the reaction time at 15 seconds. The capping reagent solution is a mixture of CapA and CapB in a molar ratio of 1:1. The molar ratio of the capping reagent to the nucleic acid sequence bound to the solid support is acetic anhydride:N-methyl The ratio of thiimidazole to the nucleic acid sequence bound to the solid support is 1:1:1.

[0491] The oxidation reaction conditions were the same for each reaction, with the temperature at 25°C, the reaction time at 15 seconds, and the concentration of the oxidation reagent at 100 ppm. 0.05M iodine in water. Iodine is bound to the solid support in the coupling step. The molar ratio of the nucleic acid sequence to the tetrahydrofuran:water:pyridine = 30:1. The experiment was carried out using a 1:1 mixed solvent.

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

[0493] After the coupling of the last nucleoside monomer is completed, the nucleic acid sequence is coupled to the solid support in order. was subjected to cleavage, deprotection, purification, desalting, and then freeze-drying to obtain the sense strand.

[0494] The cleavage and deprotection conditions are as follows: The column was then added to ammonia water with a concentration of 25 wt%, and the dose of ammonia water was 0.5 ml / μmo. The mixture was reacted at 55°C for 16 h, the remaining carrier was removed by filtration, and the supernatant was concentrated under vacuum to dryness. did.

[0495] The purification and desalting conditions were as follows: Preparative ion chromatography purification column Purification of the nucleic acids was accomplished by gradient elution with NaCl (Source 15Q). Specifically, eluent A was 20 mM sodium phosphate (pH 8.1), and the solvent was water / acetonitrile. Nitrile = 9:1 (volume ratio), eluent B: 1.5M sodium chloride, 20mM phosphorus The solvent was water / acetonitrile = 9:1 (volume ratio), Elution gradient: Eluent A:Eluent B = 100:0 to 50:50. After collecting, they are combined and desalted using a reverse phase chromatography purification column. The desalted sample was collected using a dextran gel column, and the packing material was dextran gel G25 (Se phadex G25) and was eluted with deionized water.

[0496] The detection method is as follows: Ion exchange chromatography (IEX-HPLC) The purity of the sense strand was detected using liquid chromatography-mass spectrometry (LC-MS). The molecular weight was analyzed by the method described above. The theoretical value was 7584.5, and the actual value was 7584.0. The measured value was consistent with the theoretical value, and the sense strand SS had an L-9 conjugate molecule at the 3' end. was shown to be 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 loa Deded NittoPhase® HL Solid Supports, Novate Life Sciences) is used as the starting material, which is then circulated and complexed with siRNA. The antisense strand of the L10-siXOf1M1S antibody was synthesized. protection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting The conditions were the same as those for the synthesis of the sense strand. The antisense strand AS was obtained by lyophilization.

[0498] The purity of the antisense strand was examined by ion exchange chromatography (IEX-HPLC). The molecular weight of the antisense strand was determined by liquid chromatography-mass spectrometry (LC-MS). The results showed that the measured values ​​were consistent with the theoretical values, and that the antisense strand had the target sequence. It was shown that AS was synthesized.

[0499] (1-4) Synthesis of siRNA complex L10-siXOi1M1S The sense and antisense strands of the siRNA complex L10-siXOi1M1S were Each was dissolved in water for injection to obtain a 40 mg / mL solution, mixed in an equimolar ratio, and incubated at 50°C. After heating for 15 min and cooling at room temperature, the annealed product was obtained and freeze-dried. A freeze-dried powder was obtained. Ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm( The siRNA complex was diluted to a concentration of 0.2 mg / mL using 25°C Liquid Chromatography Mass Spectrometer (LC-MS) aphy-Mass Spectrometry, purchased from Waters, model number: LC The molecular weight was detected by a FT-Premier. The measured value was consistent with the theoretical value. The siRNA complexes were determined to be double-stranded nucleic acid sequences containing L-9 complex molecules. The structure is shown in formula (403). The siRNA is selected from the group consisting of s The sequence corresponded to the iRNA complex L10-siXOi1M1S.

[0500] [Table 3] Here, capital letters C, G, U, and A represent the base sequence of a nucleotide, and lower case letter m represents the corresponding The letter m indicates that the 1 nucleotide adjacent to the left is a methoxy-modified nucleotide. The lowercase letter f indicates that the 1 nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide. The lowercase letter s indicates that the nucleotide is a thiol. It indicates that it is bonded via a phosphate ester group.

[0501] (Preparation Example 2 and Comparative Preparation Example 3) Substitution of the sense and antisense strands of the siRNA complex L10-siXOi1M1S Instead, the sense and antisense strands corresponding to L10-siXOk1M1S and NC in Table 3 The method of Preparation Example 1 was followed, except that the sense strand was used, and the siRNA complex L10 was further prepared. We synthesized siRNA complexes NC and siRNA complex NC. The siRNAs contained in the combination are L10-siXOk1M1S and N C has a sense strand and an antisense strand corresponding to C.

[0502] After the preparation is completed, the siRNA complex L10-s prepared according to the method of Preparation Example 1 is When the molecular weights of iXOk1M1S and NC were detected, the measured values ​​were consistent with the theoretical values. The synthesized siRNA complex is a double-stranded nucleic acid sequence of a specific design that contains the L-9 complex molecule. It was shown that the structures of these siRNA complexes are shown in formula (403). The siRNAs contained in each of the siRNA complexes L10-siXOk shown in Table 3 are The sequences corresponded to 1M1S and NC.

[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 by solid phase synthesis. Each was synthesized and prepared in equimolar amounts using DEPC-treated water. The sense strand and the antisense strand were dissolved, respectively, and then annealed to obtain the nucleotide sequence shown in Table 4 of the present disclosure. siXOa1M1S, siXOb1M1S, siXOc1M1S, si XOd1M1S, siXOe1M1S, siXOf1M1S, siXOg1M1S, si XOh1M1S, siXOi1M1S, siXOj1M1S, siXOk1M1S, si XOl1M1S, siXOa0, siXOe0 and siXOf0, and comparative siRNAs I got CON-siXOf.

[0504] [Table 4] Here, capital letters C, G, U, and A represent the base sequence of a nucleotide, and lower case letter m represents the corresponding The letter m indicates that the 1 nucleotide adjacent to the left is a methoxy-modified nucleotide. The lowercase letter f indicates that the 1 nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide. The lowercase letter s indicates that the nucleotide is a thiol. It indicates that it is bonded via a phosphate ester group.

[0505] During the above sequence preparation process, if the target sequence contains unmodified nucleotides, cleavage and After treatment with aqueous ammonia under deprotection conditions, the amount of single-stranded nucleic acid was 0.4 ml / μm The product was dissolved in 0.3 ml / μmol of N-methylpyrrolidone, and then added 0.3 ml / μmol of triethyl Add amine and 0.6 ml / μmol of triethylamine trihydrofluoride to the ribose The 2'-TBDMS protection was removed at .

[0506] After the preparation of the siRNA or siRNA complex is completed, it is lyophilized as a solid powder. When used, it is stored in a suitable solution such as water for injection, saline (NS), phosphate buffer (P B) or redissolve in phosphate buffer solution (PBS) or other suitable buffer to the desired concentration before use. I was able to do it.

[0507] Experimental Example 1 In vitro suppression activity of the disclosed siRNA 10% fetal bovine serum (FBS, Hyclone) and 0.2% penicillin (v / v) Streptomycin (Penicillin-Streptomycin, Gibco, HeLa cells were cultured in H-DMEM complete medium (Hyclone) supplemented with HeLa (Invitrogen). K293A cells (purchased from Nanjing Kefei Biotechnology Co., Ltd.) were incubated in a 5% CO2 / 95% air-containing solution. The cells were cultured in an incubator at 37°C.

[0508] Kumico Ui-Tei et.al., Functional dissec tion of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammali an gene silencing with significantly red uced off-target effect. Nucleic Acids Re According to the method described in , 2008.36(7), 2136-2151, the test sample was Construct plasmids and evaluate the siRNAs (siXOa0, siXOe0, siX HEK293A cells were transfected with Of0 and CON-siXOf, and duplexes were then synthesized. The expression level of the luciferase reporter gene reflects the inhibitory activity of siRNA. The specific process is as follows:

[0509] [1] Construction of test plasmids psiCHECK TM -2(Promega TM ) Using a plasmid, the target sequence, s Test plasmids containing iRNA target sequences were constructed. The sequences are shown below respectively. 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 -2 plasmid cloned into the Xho I / Not I site I trained.

[0511] [2] Transfection 8 × 10 HEK293A cells 3 Cells / well were seeded into 96-well plates and incubated for 16 h. When the cell growth density reached 70-80%, the complete H-DMEM medium in the culture well was Remove all the plates by suction and add 80 μl of Opti-MEM medium (GIBCO) to each well. The incubation was continued for 1.5 h.

[0512] For each siRNA, the corresponding test plasmid was diluted to 200 nM in DEPC-treated water. The test plasmid was diluted as a standard solution of 100 μg / μl. The concentrations (as siRNA) were 10 nM, 3 nM, and 1 nM, respectively, using EPC-treated water. A working solution of siRNA was prepared.

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

[0514] For each siRNA, 1 μl of siRNA dilution standard solution with a concentration of 3 nM was 0.05 μl of smid dilution standard solution (containing 10 ng of test plasmid) and Opti-ME A 1A2 solution containing 10 μl of M medium was prepared.

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

[0516] 0.2 μl Lipofectamine TM 2000 and 10 μl Opti-M A 1B solution containing EM medium was prepared.

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

[0518] For each siRNA, one 1B solution, one 1A1 solution, and one 1A2 solution were used. Mix 1A3 solution and 1A4 solution, incubate at room temperature for 20 min, and transfection complex 1X1, 1X2, and 1X3 were obtained, and one 1B solution was mixed with one 1C solution and incubated at room temperature. The mixture was incubated for 20 min under reduced pressure to obtain transfection complex 1X4.

[0519] For each siRNA, 20 μl of each siRNA was transferred into three culture wells. Add 1x1 of the transfection complex to each well and mix until the final siRNA concentration is approximately 0. A co-transfection mixture containing 1 nM siRNA was obtained and designated as test group 1.

[0520] For each siRNA, transfect three separate culture wells with a volume of 20 μl / well. Add 1X2 transfection complex to each well and mix until the final siRNA concentration is approximately A cotransfection mixture containing 0.03 nM siRNA was obtained and designated as test group 2. Ta.

[0521] For each siRNA, transfect three separate culture wells with a volume of 20 μl / well. Add 1X3 of each transfection complex, mix evenly, and adjust the final siRNA concentration to 0. A co-transfection mixture containing .01 nM siRNA was obtained and designated test group 3.

[0522] Add 20 μl of transfection complex 1 to three separate culture wells. X4 was added to obtain a transfection mixture without siRNA, which was used as a control group. did.

[0523] Cotransfection mixtures containing siRNA and transfection mixtures without siRNA were The transfection mixture was allowed to cotransfect in culture wells for 4 h, and then 2 100 μl of H-DMEM complete medium containing 0% FBS was added. The 96-well plate was incubated in a CO 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 Dual-Glo ( Luciferase reagent and H-DMEM complete medium (volume ratio 1: Add 1) and mix thoroughly until homogenous. Incubate at room temperature for 10 minutes, then add 120 ml of the mixture. 1 μl was transferred to a 96-well ELISA plate and assayed using the Synergy II multifunctional microplate. Read the Firefly chemiluminescence value (Fir) using a rate reader (BioTek). Add 60 μl of Dual-Glo® to each well. Stop & Gl Add o(R) reagent, mix thoroughly until homogenous, and incubate at room temperature for 10 min. According to the read-out Fir arrangement, Renill was measured using a microplate reader. The chemiluminescence value (Ren) of a was read out.

[0525] The luminescence ratio of each well, Ratio=Ren / Fir, was calculated, and the luminescence ratio of each test group or control group was Ratio(Test) or Ratio(Control) is 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, and the ratio was calculated as Ratio(Test) / R The ratio R of the control to the Renilla reporter gene expression level was obtained. , that is, the remaining activity. Inhibition rate of the target sequence = (1-R) ​​x 100%.

[0526] The results of the inhibitory activity against the target sequence by the siRNAs evaluated at different concentrations are shown in Table 5. Ta.

[0527] [Table 5]

[0528] As can be seen from the results, the siRNAs of the present disclosure were superior to the target sequence at each concentration. The inhibitory activity in vitro was also concentration-dependent. The target sequence inhibition rate was at least 61.39%, and in particular, siXOf was at a concentration of 0.0 At 3 nM, the target sequence was inhibited by 68.79%, and at a concentration of 0.1 nM, the target sequence inhibition rate was even lower. In sharp contrast, the comparative siRNA CON-s Although the sequence of iXOf is very similar to that of siXOf, it is difficult to obtain the target sequence even at a concentration of 0.1 nM. The suppression rate of XO genes was only 48.24%, which is surprising since the siRNA disclosed here has a good suppression rate. It was revealed that it had an expression-suppressing effect.

[0529] Experimental Example 2 IC of siRNA against XO mRNA in CAL-27 cells 50 Detection of 10% fetal bovine serum (FBS, Hyclone) and 0.2% penicillin (v / v) Streptomycin (Penicillin-Streptomycin, Gibco, The CA was cultured in H-DMEM complete medium (Hyclone) supplemented with 100 mM NaCl (Invitrogen). L-27 cells (purchased from Nanjing Kebei Biotechnology Co., Ltd.) were incubated in a 5% CO2 / 95% air-containing incubator. The cells were cultured in an incubator at 37°C.

[0530] 7.5 × 10 CAL-27 cells 4 Seed 16 cells / well into a 24-well plate When the cell growth density reached 70-80% after h, add H-DMEM complete medium in the culture well. Remove all of the medium by suction and add 500 μl of Opti-MEM medium (GIBCO) to each well. The cells were washed with HBSS solution, mixed uniformly, and then resuspended in 1.5 h of PBS. 6×10 5 Cells / well were inoculated into a 96-well plate, and the inoculation volume was 45 μl / well. It was El.

[0531] siXOa1M1S, siXOb1M1S, siXOc1M using DEPC-treated water 1S, siXOd1M1S, siXOe1M1S, and siXOf1M1S siRNAs The concentrations were 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, and 0.006 μM. A total of eight siRNA dilutions were used: 4 μM, 1.44 nM, and 0.72 nM (as siRNA). Standard dilution solutions were prepared for each.

[0532] The above eight types of CAL-27 cells were added to different culture wells at 15 μL / well. In this way, siRNA dilution standard solutions of different concentrations were added in sequence. The final concentrations of siRNA in each culture well were 5 μM, 1 μM, and 0.2 μM, respectively. , 0.04μM, 0.008μM, 0.0016μM, 0.32nM, 0.064nM The test group was prepared by inoculating only CAL-27 cells with siRNA dilution. Culture wells to which no standard solution was added served as controls.

[0533] Using an electroporator (EBXP-H1, Ichida Cell Electroporator) Test and control groups were electroporated and transfection parameters were: Voltage: 210V, Pulse Duration: 1 The pulse duration was set to 100 μs, pulse count was set to 6, and pulse interval was set to 1000 ms.

[0534] The above-mentioned transfected test and control samples were cultured in each well. 240 μl of H-DMEM complete medium containing 20% ​​FBS was added to each well, and transfection was performed. For each culture well, additional transfected cells were added. The cell culture medium was transferred to two culture wells of a 24-well plate, 140 μl per well. Add 855 μl of 20% FBS to each well of the 24-well plate containing cell culture medium. The complete H-DMEM medium containing IgG was added, and the culture was continued for 24 hours to obtain the test cell culture solution. , RNAVzol (Vigorous Biotechnology (Beijing) Co., Ltd. (purchased from Ito Pharmaceutical Co., Ltd., No. N002) was used to cover each well according to the procedure described in the manual. Total RNA was extracted from the test cell culture fluid.

[0535] 1 μg of total RNA was collected from each well of a 24-well plate. Goldenstar Reverse Transcription Kit TM RT6 cDNA Synthesis The kit was purchased from Beijing Keike Xinye Biotechnology Co., Ltd., No. TSK301M. From the reagents, Goldenstar TM Oligo (dT) 17 Select the primer Then, 20 μl of the reverse transcription reaction system was arranged according to the reverse transcription procedure in the kit's manual. The reverse transcription was performed on the total RNA of the cells. After incubation at 85°C for 50 min, then at 85°C for 5 min, and finally at 4°C for 30 s, the reaction was completed. Then, 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. , NovoStart® SYBR qPCR SuperMix Plus Reagents provided by the kit (purchased from Kinki Protein Technology Co., Ltd., No. E096-01B) A 20 μl qPCR reaction was set up using the following: The PCR primer sequences for amplifying the gene GAPDH are shown in Table 7. The final concentration of each primer was 0.25 μM. Each qPCR reaction was performed using ABI Step Place the PCR product in the OnePlus Real-Time PCR machine and amplify it in a three-step process. The procedure consisted of 10 min pre-denaturation at 95°C, 30 s denaturation at 95°C, and 30 s annealing at 60°C. The denaturation, annealing, and extension were repeated a total of 40 times. After this, product W was obtained, which contained the amplified target gene XO and the endogenous reference gene GAPDH. Then, product W was incubated at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s in sequence. The target genes XO and XO in product W were detected by real-time fluorescent quantitative PCR. The melting curves of the target gene XO and the endogenous reference gene GAPDH were obtained, and the melting curves of the target gene XO and the endogenous reference gene GAPDH were obtained. The Ct values ​​of APDH were obtained.

[0537] [Table 6]

[0538] Using the comparative Ct (ΔΔCt) method, the expression level of the target gene XO in each test group and the control group was Relative quantitative calculation was performed for the following: 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 (average of the control group) is the ΔCt of each of the two culture wells in the control group. t (control group). Therefore, each culture well of the test group and the control group Each corresponds to one ΔΔCt value.

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

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

[0541] The relative expression level of XO mRNA in each test group at each concentration for siRNA of the same test group The mean of each well is the arithmetic mean of the relative expression levels of duplicate culture wells at that concentration.

[0542] Graphpad 6.0 software log(inhibitor) vs. re The response-variable slope function allows fitting of the dose-effect curve. The IC value of each siRNA against XO mRNA was calculated from the dose-effect curve. 50 Calculate the value Specifically, the fitted dose-effect curve satisfies the following formula:

[0543]

number

[0544] Figures 1A to 1F show, in order, siXOa1M1S, siXOb1M1S, and siXOc1M1S. , siXOd1M1S, siXOe1M1S and siXOf1M1S were transfected. After screening, the results were analyzed according to the relative expression levels of XO mRNA in CAL-27 cells in vitro. From this dose-effect curve and the corresponding formula, Y = 50% when 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 inhibited the expression of CAL-27 cells in vitro. It showed high XO mRNA suppression activity in IC 50 is 0.037 to 0.3277 μM It was.

[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 and cultured using type I collagen. Mouse primary hepatocytes were seeded onto tissue culture dishes coated with 1× penicillin-streptomycin. The cells were incubated in RPMI 1460 medium containing ampicillin and 10% FBS under 5% CO2 / 95% air. The cells were cultured in an air-containing incubator at 37°C for 30 min.

[0549] Discard the medium and culture the primary mouse hepatocytes in opti-MEM at a density of 1 × 10 6 Cells / mL The suspension of mouse primary hepatocytes was then prepared by adjusting the amount of the culture medium. The obtained mouse primary hepatocyte suspension was added to each of the culture wells, and the mouse primary hepatocytes were cultured. The volume of mouse primary hepatocyte suspension added was 0.5 mL / well, and the mouse The number of primary hepatocytes was 5 × 10 4 cells / well.

[0550] siXOg1M1S, siXOh1M1S, siXOi1M1 with DEPC-treated water S, siXOj1M1S, siXOk1M1S or siXOl1M1S siRNA , and 20 μM siRNA standard diluted solutions were prepared.

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

[0552] Prepare 1B solution. Add 1 μl of Lipofectamine to each 1B solution. TM 200 0 and 50 μl of Opti-MEM medium.

[0553] Mix one of the 1B solutions with the 1A solution of each siRNA obtained, and incubate at room temperature. After culturing for 20 minutes, 1X transfection complexes of each siRNA were obtained.

[0554] Mix one 1B solution with 50 μl of Opti-MEM medium and incubate at room temperature for 20 min. Transfection complex 1X' was obtained.

[0555] Each siRNA was transfected into the culture wells at a volume of 100 μl / well. Add 1X siRNA complex and mix thoroughly until the final concentration of each siRNA is approximately 20 nM. The transfection complexes were obtained as follows: Transfect three culture wells each with X and inject the siRNA-containing transfectants. The resulting injection mixture was used as the test group.

[0556] Three separate culture wells were transfected with 100 μl of each solution. siRNA-free transfection mixture was obtained by adding 1X' siRNA complex. The control group ...

Claims

1. A siRNA comprising a sense strand and an antisense strand, each independently being a modified or unmodified nucleotide, and the sense strand is the antisense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, I and the nucleotide sequence II form a double-stranded region in at least a portion of a reverse complementary manner; The nucleotide sequence I and the nucleotide sequence II are shown in the following i) to xii). and one selected from the sequences listed below: i) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length; and preferably has no more than three nucleotide differences, and is shown in nucleotide sequence II and SEQ ID NO:

2. The nucleotide sequences to be compared are of equal 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 To The corresponding nucleotide Z ３ and in said nucleotide sequence II, position Z ２ Corresponding to Nucleotide Z ４ and Z ４ The first nucleotide at the 5' end of the antisense strand is It is a nucleotide, ii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61 have a length and the nucleotide difference between said nucleotide sequence II and SEQ ID NO:6 is 3 or less. The nucleotide sequence shown in FIG. 2 is the same length and has 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 To The corresponding nucleotide Z ７ and in said nucleotide sequence II, position Z ６ Corresponding to Nucleotide Z ８ and Z ８ The first nucleotide at the 5' end of the antisense strand is It is a nucleotide, iii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 are The nucleotide sequence is the same in length and has no more than three nucleotide differences, and is identical to the nucleotide sequence II of SEQ ID NO: The nucleotide sequence shown in No. 122 is the same length and has no more than three nucleotide differences. and 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 To The corresponding nucleotide Z １２ and Z １２ is the 5' end of the antisense strand It is the first nucleotide, iv) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 have a length and the nucleotide sequence II and the sequence ID No. The nucleotide sequence shown in 182 is identical in length and has no more than three nucleotide differences. can be, 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 １ ４ Nucleotide Z corresponding to １６ and Z １６ is the 5' end of the antisense strand It is the first nucleotide at the end v) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 241 have a length and the nucleotide difference between said nucleotide sequence II and SEQ ID NO:2 is 3 or less. The nucleotide sequence shown in 42 is identical in length and has no more than three nucleotide differences. the law of nature, 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 １ ８ Nucleotide Z corresponding to ２０ and Z ２０ is the 5' end of the antisense strand It is the first nucleotide at the end vi) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 have a length and the nucleotide sequence II and the sequence ID No. The nucleotide sequence shown in 302 is the same length and has no more than three nucleotide differences. can be, 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 ２ ２ Nucleotide Z corresponding to ２４ and Z ２４ is the 5' end of the antisense strand It is the first nucleotide at the end vii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 are The nucleotide sequence is the same in length and has no more than three nucleotide differences, and is identical to the nucleotide sequence II of SEQ ID NO: The nucleotide sequence shown in No. 362 is identical in length and has no more than three nucleotide differences. and 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 ２ ６ Nucleotide Z corresponding to ２８ and Z ２８ is the 5' end of the antisense strand It is the first nucleotide at the end viii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 421 , which are equal in length and have no more than three nucleotide differences, and which are identical to nucleotide sequence II and sequence The nucleotide sequence shown in FIG. 422 is the same length and has three or more nucleotide differences. Below, 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 ３ ０ Nucleotide Z corresponding to ３２ and Z ３２ is the 5' end of the antisense strand It is the first nucleotide at the end ix) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 481 have a length and the nucleotide sequence II and the sequence ID No. The nucleotide sequence shown in 482 is identical in length and has no more than three nucleotide differences. can be, 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 ３ ４ Nucleotide Z corresponding to ３６ and Z ３６ is the 5' end of the antisense strand It is the first nucleotide at the end x) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:541 have a length and the nucleotide difference between said nucleotide sequence II and SEQ ID NO:5 is 3 or less. The nucleotide sequence shown in 42 is identical in length and has no more than three nucleotide differences. the law of nature, 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 ３ ８ Nucleotide Z corresponding to ４０ and Z ４０ is the 5' end of the antisense strand It is the first nucleotide at the end xi) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 601 have a length and the nucleotide sequence II and the sequence ID No. The nucleotide sequence shown in 602 is the same length and has no more than three nucleotide differences. can be, 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 ４ ２ Nucleotide Z corresponding to ４４ and Z ４４ is the 5' end of the antisense strand It is the first nucleotide at the end xii) the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:661 are The nucleotide sequence is the same in length and has no more than three nucleotide differences, and is identical to the nucleotide sequence II of SEQ ID NO: The nucleotide sequence shown in No. 662 is identical in length and has no more than three nucleotide differences. and 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 ４ ６ Nucleotide Z corresponding to ４８ and Z ４８ is the 5' end of the antisense strand The first nucleotide at the end of the siRNA.

2. There is a nucleotide between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:

1. and / or the nucleotide sequence II has no more than one amino acid difference with the nucleotide sequence shown in SEQ ID NO:

2. There is no more than one nucleotide difference between the nucleotide sequence, Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61 and / or said nucleotide sequence II and SEQ ID NO:6 have no more than one nucleotide difference. 2 has no more than one nucleotide difference between it and the nucleotide sequence shown in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:121 and / or said nucleotide sequence II and SEQ ID NO: 122 has no more than one nucleotide difference between it and the nucleotide sequence shown in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:181 and / or said nucleotide sequence II and SEQ ID NO: 182 has no more than one nucleotide difference between it and the nucleotide sequence shown in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:241 and / or said nucleotide sequence II and SEQ ID NO: 242 has no more than one nucleotide difference between it and the nucleotide sequence shown in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:301 and / or said nucleotide sequence II and SEQ ID NO: 302 has no more than one nucleotide difference from the nucleotide sequence set forth in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:361 and / or said nucleotide sequence II and SEQ ID NO: 362 has no more than one nucleotide difference from the nucleotide sequence set forth in Alternatively, between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:421 and / or said nucleotide sequence II and SEQ ID NO: 422 has no more than one nucleotide difference between it and the nucleotide sequence set forth in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:481 and / or said nucleotide sequence II and SEQ ID NO: 482 has no more than one nucleotide difference between it and the nucleotide sequence shown in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:541 and / or said nucleotide sequence II and SEQ ID NO: 542 has no more than one nucleotide difference between it and the nucleotide sequence set forth in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:601 and / or said nucleotide sequence II and SEQ ID NO: 602 has no more than one nucleotide difference from the nucleotide sequence shown in Alternatively, between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:661 and / or said nucleotide sequence II and SEQ ID NO: 662 has no more than one nucleotide difference between it and the nucleotide sequence shown in 1. siRNA described in

3. The nucleotide sequence between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 The difference between the chids is Z ４ Including the difference at the position of Z ４ is selected from A, C or G; Alternatively, between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:62 The nucleotide difference is Z ８ Including the difference at the position of Z ８ is selected from U, C or G; Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 The nucleotide difference between Z １２ Including the difference at the position of Z １２ is selected from U, C or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 The nucleotide difference between Z １６ Including the difference at the position of Z １６ is selected from U, C or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 The nucleotide difference between Z ２０ Including the difference at the position of Z ２０ Select from A, C or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 302 The nucleotide difference between Z ２４ Including the difference at the position of Z ２４ Select from A, C or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 362 The nucleotide difference between Z ２８ Including the difference at the position of Z ２８ is selected from A, U or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 422 The nucleotide difference between Z ３２ Including the difference at the position of Z ３２ Select from A, C or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 482 The nucleotide difference between Z ３６ Including the difference at the position of Z ３６ is selected from U, C or G And, Alternatively, the nucleotide sequence II may be a nucleotide sequence as set forth in SEQ ID NO:

542. The nucleotide difference between Z ４０ Including the difference at the position of Z ４０ Select from A, C or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:602 The nucleotide difference between Z ４４ Including the difference at the position of Z ４４ is selected from U, C or G And, Alternatively, the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:662 The nucleotide difference between Z ４８ Including the difference at the position of Z ４８ is selected from U, C or G The siRNA according to claim 1 or 2.

4. Z ３ Is, Z ４ or Z ７ Is, Z ８ and complementary nuclease or Z １１ Is, Z １２ or Z １ ５ Is, Z １６ or Z １９ Is, Z ２０ and complementary Nuk or Z ２３ Is, Z ２４ or Z ２７ Is, Z ２８ or Z ３１ Is, Z ３２ and complementary or Z ３５ Is, Z ３６ or Z ３９ Is, Z ４０ or Z ４３ Is, Z ４４ Complementary to nucleotide, or Z ４７ Is, Z ４８ and wherein the nucleotide is complementary to 4. The siRNA according to any one of 1 to 3.

5. The sense strand and the antisense strand may have the same or different lengths, and the length of the sense strand is 19 23 nucleotides in length and the antisense strand is 19-26 nucleotides in length; The nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:3, nucleotide sequence II is the 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 and complementary nucleotide It is Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

63. wherein the 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, Z ８ is selected from A, U, G or C; Z ７ Is, Z ８ and complementary nucleotide It is Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

123. and said nucleotide sequence II is the nucleotide sequence set forth 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 and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

183. and said 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 AUACUCUGAGAGAGAGAUCC-3 '(SEQ ID NO: 184) However, Z １６ is selected from A, U, G or C; Z １５ Is, Z １６ and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

243. and said 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, Z ２０ is selected from A, U, G or C; Z １９ Is, Z ２０ and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

303. and said 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, Z ２４ is selected from A, U, G or C; Z ２３ Is, Z ２４ and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

363. and said 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 and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown 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, Z ３２ is selected from A, U, G or C; Z ３１ Is, Z ３２ and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

483. and said 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 and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

543. and said 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 and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

603. and said 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, Z ４４ is selected from A, U, G or C; Z ４３ Is, Z ４４ and complementary Nuk It is leotide, Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:

663. and said 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 and complementary Nuk The siRNA according to any one of claims 1 to 4, which is a leucine.

6. Z ３ is A and Z ４ is U, or Z ７ is U and Z ８ is A, or , Z １１ is U and Z １２ is A, or Z １５ is U and Z １６ is A , or Z １９ is A and Z ２０ is U, or Z ２３ is A and Z ２４ teeth U, or Z ２７ is G and Z ２８ is C; or Z ３１ is A, Z ３２ is U, or Z ３５ is U and Z ３６ is A, or Z ３９ A And Z ４０ is U, or Z ４３ is U and Z ４４ is A, or Z ４７ is U and Z ４８ The siRNA of claim 5, wherein is A.

7. The sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence III. The length of nucleotide sequence III and nucleotide sequence IV is are each independently 1 to 4 nucleotides, and said nucleotide sequence III is a nucleotide nucleotide sequence I is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. The nucleotide sequence III and the nucleotide sequence IV are equal in length. The term "substantially reverse complementary" refers to a combination of two This refers to the presence of one or fewer base mismatches between nucleotide sequences, making it completely reverse-complementary. Any of claims 1 to 6, wherein "no mismatch" means that there is no mismatch between the two nucleotide sequences.

1. An siRNA according to claim 1.

8. The nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1 are equal in length. The nucleotide differences are three or less, and the nucleotide sequences III and IV are short in length. each of the nucleotides is a single nucleotide, and the base of the nucleotide sequence III is U, or The nucleotide sequences III and IV are both 2 nucleotides in length and each have a 5' end beginning with from the 3' end of nucleotide sequence III to the 3' end of nucleotide sequence III is UU; or Nucleotide sequences III and IV are each 3 nucleotides in length and are located 3 nucleotides from the 5' end. Towards the 'end, the base sequence of nucleotide sequence III is AUU, or The nucleic acid sequences III and IV are both 4 nucleotides in length and extend from the 5' end to the 3' end. Towards the end, the base sequence of nucleotide sequence III is CAUU; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61 have a length and the nucleotide sequences III and IV are of equal length and have no more than three nucleotide differences. , each of which is 1 nucleotide in length, and the base of said nucleotide sequence III is A; or 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 AA; and The nucleotide sequences III and IV are each 3 nucleotides in length and 5' From the terminus towards the 3' terminus, the base sequence of nucleotide sequence III is UAA; or The nucleotide sequences III and IV are each 4 nucleotides in length and each have a 5' end From the 3' end, the base sequence of nucleotide sequence III is GUAA; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 121 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is C; or 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 GC; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is AGC; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminus towards the 3' terminus, the base sequence of nucleotide sequence III is CAGC; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is A; or 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 CA; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is CCA; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminal to the 3' terminal, the base sequence of nucleotide sequence III is CCCA; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:241 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is C; or 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 CC; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is UCC; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminal to the 3' terminal, the base sequence of nucleotide sequence III is UUCC; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is C; or 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 CC; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is GCC; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminal to the 3' terminal, the base sequence of nucleotide sequence III is UGCC; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 361 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is G; or 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 GG; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is AGG; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminus towards the 3' terminus, the base sequence of nucleotide sequence III is AAGG; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:421 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is U; or 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; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is GGU; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminus towards the 3' terminus, the base sequence of nucleotide sequence III is GGGU; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:481 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is G; or 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 AG; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is GAG; The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminal to the 3' terminal, the base sequence of nucleotide sequence III is UGAG; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:541 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is A; or 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; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is AAA; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminus towards the 3' terminus, the base sequence of nucleotide sequence III is UAAA; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:601 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is G; or 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 GG; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is UGG; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminus towards the 3' terminus, the base sequence of nucleotide sequence III is GUGG; Alternatively, the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:661 are The nucleotide sequences III and IV are equal in length and have no more than three nucleotide differences. each of which is one nucleotide in length, and the base of said nucleotide sequence III is U; or 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 AU; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length and From the 3' end to the 3' end, the base sequence of nucleotide sequence III is UAU; and The nucleotide sequences III and IV are each 4 nucleotides in length and 5' From the terminal to the 3' terminal, the base sequence of nucleotide sequence III is CUAU. The siRNA described in claim 7.

9. The antisense strand further comprises a nucleotide sequence V, the nucleotide sequence V having a length The length of the antisense strand is 1 to 3 nucleotides, and the antisense strand is bound to the 3' end of the antisense strand. The siRNA according to any one of claims 1 to 8, wherein the 3'-overhanging end of the siRNA strand is a 3'-overhanging end of the siRNA strand.

10. The siRNA of claim 9, wherein the nucleotide sequence V is 2 nucleotides in length. 。

11. The nucleotide sequence V is a sequence of two consecutive thymine deoxyribonucleotides or or the nucleotide sequence V is a target The si gene of claim 9 or 10 is complementary to a nucleotide at a corresponding position in mRNA. RNA.

12. The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:5, The antisense strand comprises the nucleotide sequence set forth 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. wherein 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 shown in SEQ ID NO:

65. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

66. fruit, 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 shown in SEQ ID NO:

67. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

68. fruit, 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 shown in SEQ ID NO:

125. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

126. Including, 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 shown in SEQ ID NO:

127. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

128. Including, 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

185. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

186. Including, 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 shown in SEQ ID NO:

187. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

188. Including, 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

245. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

246. Including, 5'-ACAUGGACAACUGCUAUAZ １９ -3' (SEQ ID NO: 245), 5'-Z ２０ UAUAGCAGUUGUCCAUGUGG-3 '(SEQ ID NO: 246) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

247. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

248. Including, 5'-CCACAUGGACAACUGCUAUAZ １９ -3' (SEQ ID NO: 247), 5'-Z ２０ UAUAGCAGUUGUCCAUGUGGAA-3' (SEQ ID NO: 248 ） However, the Z １６ 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

305. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

306. Including, 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 shown in SEQ ID NO:

307. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

308. Including, 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

365. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

366. Including, 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 shown in SEQ ID NO:

367. wherein 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

425. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

426. Including, 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 shown in SEQ ID NO:

427. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

428. Including, 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

485. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

486. Including, 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 shown in SEQ ID NO:

487. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

488. Including, 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

545. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

546. Including, 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 shown in SEQ ID NO:

547. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

548. Including, 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

605. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

606. Including, 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 shown in SEQ ID NO:

607. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

608. Including, 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 the law of nature, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

665. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

666. Including, 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 shown in SEQ ID NO:

667. wherein the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

668. Including, 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 is a nucleotide complementary to The siRNA according to any one of claims 1 to 11.

13. siXOa1, siXOa2, siXOb1, siXOb2, siXOc1, siXO c2, siXOd1, siXOd2, siXOe1, siXOe2, siXOf1, si XOf2, siXOg1, siXOg2, siXOh1, siXOh2, siXOi1, siXOi2, siXOj1, siXOj2, siXOk1, siXOk2, siXOl siXO11 and siXO12 according to any one of claims 1 to 12. RNA.

14. The sense strand or the antisense strand has at least one modified nucleotide. and / or at least one phosphate ester group is a phosphate having a modifying group. The siRNA according to any one of claims 1 to 13, which is an ester group.

15. Each nucleotide in the sense strand and the antisense strand is independently fluoro-modified.

15. The method according to claim 1, wherein the nucleotide is a nucleotide or a non-fluoro modified nucleotide. The siRNA described herein.

16. The fluoro-modified nucleotides are located at nucleotide sequence I and nucleotide sequence II. from the 5' end to the 3' end, at least the 7th, 8th, The 9th nucleotide is a fluoro-modified nucleotide, and from the 5' to the 3' end At least the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are The siRNA of claim 15, which is a fluoro-modified nucleotide.

17. From the 5' end to the 3' end, in the sense strand, The nucleotides at positions 7, 8, and 9 or positions 5, 7, 8, and 9 are fluoro-modified nucleotides. and the nucleotides at the remaining positions in the sense strand are non-fluoro modified nucleotides. From the 5' end to the 3' end, in the antisense strand, The nucleotides at positions 2, 6, 14, and 16 or 2, 6, 8, 9, 14, and 16 of column II are and the nucleotides at the remaining positions in the antisense strand are fluoro-modified nucleotides. The siRNA of claim 16, wherein is a non-fluoro modified nucleotide.

18. Each non-fluoro modified nucleotide has a hydroxy group at the 2' position of the ribose group of the nucleotide. are independently selected from nucleotides or nucleotide analogs substituted with non-fluorine groups. The siRNA according to any one of claims 15 to 17, which is one of the above.

19. A nucleoside in which the 2'-hydroxyl group of the ribose group of a nucleotide is replaced with a non-fluorine group. 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-Alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino Modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxy nucleotides and the nucleotide analog is an isonucleotide, an LNA, an ENA 19. The siRNA of claim 18, which is one selected from the group consisting of cET, UNA and GNA. 。

20. Each non-fluoro modified nucleotide is a methoxy modified nucleotide, Toxo-modified nucleotides are nucleotides in which the 2'-hydroxy group of the ribose group is replaced with methoxy. The siRNA of any one of claims 15 to 19, which refers to a nucleotide.

21. From the 5' end to the 3' end, the nucleotide sequence in the sense strand of the siRNA The nucleotides at positions 5, 7, 8 and 9 of column I are fluoro-modified nucleotides; The remaining nucleotides in the sense strand of the nucleic acid are methoxy-modified nucleotides, and the 5' end From the 3' end to the nucleotide sequence in the antisense strand of the siRNA The nucleotides at positions 2, 6, 8, 9, 14, and 16 of II are fluoro-modified nucleotides. and the remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides. Chid, Alternatively, the nucleotide sequence of the sense strand of the siRNA from the 5' end to the 3' end may be The nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I 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' end to the 3' end, The nucleotides at positions 2, 6, 14, and 16 of peptide sequence II are fluoro-modified nucleotides. and the remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides. Chid, Alternatively, the nucleotide sequence of the sense strand of the siRNA from the 5' end to the 3' end may be The nucleotides at positions 7, 8 and 9 of nucleotide sequence I are fluoro-modified nucleotides; The nucleotides at the remaining positions of the sense strand of the iRNA are methoxy-modified nucleotides, nucleotides in the antisense strand of the siRNA from the 3' end to the 3' end The nucleotides at positions 2, 6, 14, and 16 of sequence II are fluoro-modified nucleotides. , the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides The siRNA of claim 17,

22. 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, siXOc2-M1 , siXOc2-M2, siXOc2-M3, siXOd1-M1, siXOd1-M2 , siXOd1-M3, siXOd2-M1, siXOd2-M2, siXOd2-M3 , siXOe1-M1, siXOe1-M2, siXOe1-M3, siXOe2-M1 , siXOe2-M2, siXOe2-M3, siXOf1-M1, siXOf1-M2 , siXOf1-M3, siXOf2-M1, siXOf2-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, siXOk2-M1 , siXOk2-M2, siXOk2-M3, siXOl1-M1, siXOl1-M2 , siXOl1-M3, siXOl2-M1, siXOl2-M2 and siXOl2-M The siRNA according to any one of claims 1 to 21, which is any one of the above 3.

23. The phosphate ester group having the modifying group is a phosphate diester bond of the phosphate ester group. wherein at least one oxygen atom of the formula (I) is replaced with a sulfur atom. Item 15. The siRNA described in item 14.

24. The phosphate ester group having the modifying group is a thiophosphoric acid having a structure represented by formula (1). The siRNA of claim 14 or 23, which is an ester group. 【Chemistry 1】

25. In the siRNA, the thiophosphate group is 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, 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 Between, and The second and third nucleotides from the 3' end of the antisense strand The si according to claim 22 or 23, wherein the si is bound to at least one of the group consisting of RNA.

26. siXOa1-M1S, siXOa1-M2S, siXOa1-M3S, siXOa2 -M1S, siXOa2-M2S, siXOa2-M3S, siXOb1-M1S, si XOb1-M2S, siXOb1-M3S, siXOb2-M1S, siXOb2-M2 S, siXOb2-M3S, siXOc1-M1S, siXOc1-M2S, siXOc 1-M3S, siXOc2-M1S, siXOc2-M2S, siXOc2-M3S, s iXOd1-M1S, siXOd1-M2S, siXOd1-M3S, siXOd2-M 1S, siXOd2-M2S, siXOd2-M3S, siXOe1-M1S, siXO e1-M2S, siXOe1-M3S, siXOe2-M1S, siXOe2-M2S, siXOe2-M3S, siXOf1-M1S, siXOf1-M2S, siXOf1- M3S, siXOf2-M1S, siXOf2-M2S, siXOf2-M3S, siX Og1-M1S, siXOg1-M2S, siXOg1-M3S, siXOg2-M1S , siXOg2-M2S, siXOg2-M3S, siXOh1-M1S, siXOh1 -M2S, siXOh1-M3S, siXOh2-M1S, siXOh2-M2S, si XOh2-M3S, siXOi1-M1S, siXOi1-M2S, siXOi1-M3 S, siXOi2-M1S, siXOi2-M2S, siXOi2-M3S, siXOj 1-M1S, siXOj1-M2S, siXOj1-M3S, siXOj2-M1S, s iXOj2-M2S, siXOj2-M3S, siXOk1-M1S, siXOk1-M 2S, siXOk1-M3S, siXOk2-M1S, siXOk2-M2S, siXO k2-M3S, siXOl1-M1S, siXOl1-M2S, siXOl1-M3S, Any one of siXOl2-M1S, siXOl2-M2 and siXOl2-M3S The siRNA according to any one of claims 1 to 25.

27. siXOa1-M1P1, siXOa1-M2P1, siXOa1-M3P1, siX Oa2-M1P1, siXOa2-M2P1, siXOa2-M3P1, siXOa1- M1SP1, siXOa1-M2SP1, siXOa1-M3SP1, siXOa2-M 1SP1, siXOa2-M2SP1, siXOa2-M3SP1, siXOb1-M1 P1, siXOb1-M2P1, siXOb1-M3P1, siXOb2-M1P1, s iXOb2-M2P1, siXOb2-M3P1, siXOb1-M1SP1, siXO b1-M2SP1, siXOb1-M3SP1, siXOb2-M1SP1, siXOb 2-M2SP1, siXOb2-M3SP1, siXOc1-M1P1, siXOc1- M2P1, siXOc1-M3P1, siXOc2-M1P1, siXOc2-M2P1 , siXOc2-M3P1, siXOc1-M1SP1, siXOc1-M2SP1, s iXOc1-M3SP1, siXOc2-M1SP1, siXOc2-M2SP1, si XOc2-M3SP1, siXOd1-M1P1, siXOd1-M2P1, siXOd 1-M3P1、siXOd2-M1P1、siXOd2-M2P1、siXOd2-M3 P1、siXOd1-M1SP1、siXOd1-M2SP1、siXOd1-M3SP 1、siXOd2-M1SP1、siXOd2-M2SP1、siXOd2-M3SP1 、siXOe1-M1P1、siXOe1-M2P1、siXOe1-M3P1、siX Oe2-M1P1、siXOe2-M2P1、siXOe2-M3P1、siXOe1- M1SP1、siXOe1-M2SP1、siXOe1-M3SP1、siXOe2-M 1SP1、siXOe2-M2SP1、siXOe2-M3SP1、siXOf1-M1 P1、siXOf1-M2P1、siXOf1-M3P1、siXOf2-M1P1、s iXOf2-M2P1、siXOf2-M3P1、siXOf1-M1SP1、siXO f1-M2SP1、siXOf1-M3SP1、siXOf2-M1SP1、siXOf 2-M2SP1、siXOf2-M3SP1、siXOg1-M1P1、siXOg1- M2P1、siXOg1-M3P1、siXOg2-M1P1、siXOg2-M2P1 、siXOg2-M3P1、siXOg1-M1SP1、siXOg1-M2SP1、s iXOg1-M3SP1、siXOg2-M1SP1、siXOg2-M2SP1、si XOg2-M3SP1、siXOh1-M1P1、siXOh1-M2P1、siXOh 1-M3P1、siXOh2-M1P1、siXOh2-M2P1、siXOh2-M3 P1、siXOh1-M1SP1、siXOh1-M2SP1、siXOh1-M3SP 1、siXOh2-M1SP1、siXOh2-M2SP1、siXOh2-M3SP1 、siXOi1-M1P1、siXOi1-M2P1、siXOi1-M3P1、siX Oi2-M1P1、siXOi2-M2P1、siXOi2-M3P1、siXOi1- M1SP1、siXOi1-M2SP1、siXOi1-M3SP1、siXOi2-M 1SP1、siXOi2-M2SP1、siXOi2-M3SP1、siXOj1-M1 P1、siXOj1-M2P1、siXOj1-M3P1、siXOj2-M1P1、s iXOj2-M2P1、siXOj2-M3P1、siXOk1-M1P1、siXOk 1-M2P1、siXOk1-M3P1、siXOk2-M1P1、siXOk2-M2 P1, siXOk2-M3P1, siXOl1-M1P1, siXOl1-M2P1, s iXOl1-M3P1, siXOl2-M1P1, siXOl2-M2P1, siXOl 2-M3P1, siXOj1-M1SP1, siXOj1-M2SP1, siXOj1- M3SP1, siXOj2-M1SP1, siXOj2-M2SP1, siXOj2-M 3SP1, siXOk1-M1SP1, siXOk1-M2SP1, siXOk1-M3 SP1, siXOk2-M1SP1, siXOk2-M2SP1, siXOk2-M3S P1, siXOl1-M1SP1, siXOl1-M2SP1, siXOl1-M3SP 1. siXOl2-M1SP1, siXOl2-M2SP1 and siXOl2-M3SP The siRNA of any one of claims 1 to 26, selected from the group consisting of 1.

28. A composition comprising the siRNA of any one of claims 1 to 27 and a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising:

29. The weight ratio of the siRNA to the pharma- ceutical acceptable carrier is 1:(1-500). Item 29. The pharmaceutical composition according to item 28.

30. The weight ratio of the siRNA to the pharma- ceutically acceptable carrier is 1:(1-50).

29. The siRNA described in 29.

31. The pharma- ceutical acceptable carrier comprises an organic amine, a co-lipid and a PEGylated lipid, The functional amine is a compound represented by formula (201) and / or a pharma- ceutically acceptable salt thereof; 【Chemistry 2】 During the ceremony, Each X １０１ and X １０２ are each independently O, S, N-A or C-A, and A is water. Element or C １ -C ２０ is a hydrocarbon chain, Each Y １０１ and 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 and R １０７ That each independently represents hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a ring or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic groups of the formula , branched or straight chain acyl groups, substituted or unsubstituted branched or straight chain aryl groups, substituted or unsubstituted a substituted, branched or straight chain heteroaryl group; x is an integer from 1 to 10; n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1, If m=p=0, then R １０２ is hydrogen, When at least one of n or m is 2, R １０３ and nitrogen in formula (201) , forming a structure represented by formula (202) or formula (203), 【Chemistry 3】 In the formula, g, e, and f are each independently an integer from 1 to 6, and "HCC" represents a hydrocarbon chain. and each ＊ Any of claims 26 to 30, wherein N represents a nitrogen atom in formula (201). The pharmaceutical composition according to any one of claims 1 to 5.

32. The organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215). It is an organic amine that is 【Chemistry 4】 The co-lipid may be cholesterol, a cholesterol analogue, and / or a cholesterol It is a derivative of The PEGylated lipid is 1,2-dipalmitamide-sn-glycero-3-phosphatidylcholine. ethanolamine-N-[methoxy(polyethylene glycol)]-2000, 32. The pharmaceutical composition of claim 31.

33. The molar ratio of the organic amine, the co-lipid, and the PEGylated lipid is (19.7 to 80): The pharmaceutical composition according to claim 31 or 32, wherein the ratio is (19.7-80):(0.3-50). 。

34. The molar ratio of the organic amine, the co-lipid, and the PEGylated lipid is (50 to 70):(2 34. The pharmaceutical composition of claim 33, wherein the molar ratio is 0-40):(3-20).

35. siRNA according to any one of claims 1 to 27 and a compound that binds to the siRNA The siRNA conjugate comprises a conjugated group.

36. The conjugate group comprises a pharma- ceutical acceptable targeting group and a linker, 36. The method of claim 35, wherein the anchor and the targeting group are in turn covalently or non-covalently linked. The siRNA complexes shown above.

37. The linker has a structure shown in formula (301): 【Chemistry 5】 In the formula, k is an integer from 1 to 3, L Ａ is a chain portion containing an amide bond having a structure represented by formula (302), L Ａ At each end of the group, there is one target group and one L Ｃ Bonded to the part by ether bond And, 【Chemistry 6】 L Ｂ is a chain portion containing an N-acylpyrrolidine having a structure represented by formula (303). The chain portion has a carbonyl group at one end thereof, and the L Ｃ Bonded to the part by an amide bond and having an oxygen atom at the other end and being bound to the siRNA via a phosphate bond; 【Chemistry 7】 L Ｃ Hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane is a divalent to tetravalent linker group based on hydroxymethylaminomethane, Ｃ But oxygen Each of the L's is bonded to an ether bond via a Ａ is bonded to the a moiety through a nitrogen atom The L Ｂ The siRNA conjugate of claim 36 , wherein the siRNA conjugate is linked to the moiety.

38. The siRNA conjugate has the structure shown in formula (305): 【Chemistry 8】 The double helix structure represents the siRNA according to any one of claims 35 to 37. The siRNA complexes shown above.

39. The linker has a structure shown in formula (306): 【Chemistry 9】 In the formula, 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 at which the linker is linked to the siRNA via a phosphate bond. The siRNA complex of claim 38.

40. The siRNA conjugate has the structure shown in formula (307): 【Chemistry 10】 Any of claims 35, 36 and 39, wherein the double helix structure represents the siRNA.

2. The siRNA complex described in claim 1.

41. The linker is attached to the 3' end of the sense strand of the siRNA.

13. The siRNA complex according to any one of claims 1 to 12.

42. The siRNA conjugate has the structure shown in formula (308): 【Chemistry 11】 During the ceremony, n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4. 、 each m1, m2, and m3 is independently an integer selected from 2 to 10; R １０ , R １１ , R １２ , R １３ , R １４ and R １５ are each independently H; , or C １ -C １０ Alkyl group, C １ -C １０ Halogenated alkyl group and C １ -C １０ a alkoxy groups; R ３ is a group having a structure represented by formula A59, 【Chemistry 12】 In the formula, E １ is OH, SH or BH ２ and Nu is any one of claims 1 to 120. is an siRNA as described herein, R ２ is a linear alkylene group of 1 to 20 carbon atoms in length, where one or more carbon Atoms are 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 Heterocycli Rene group and C ５ -C １０ one or more selected from the group consisting of heteroarylene groups; is replaced by R ２ But, C １ -C １０ Alkyl group, C ６ -C １０ Aryl group, C ５ -C １０ Heteroaryl group, C １ -C １０ Halogenated alkyl group, -OC １ -C １０ Alkyl groups, -OC １ -C １０ Alkylphenyl group, -C １ -C １０ Alkyl-OH, -OC １ -C １ ０ Halogenated alkyl group, -SC １ -C １０ Alkyl group, -SC １ -C １０ Alkylphe Nyl group, -C １ -C １０ Alkyl-SH, -SC １ -C １０ Halogenated alkyl groups, halo -aryl 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 -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 N.H. 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 (halogenated alkyl group) may optionally have one or more substituents, Each L １ are independently a linear alkylene group of 1 to 70 carbon atoms in length, Multiple carbon atoms are C(O), NH, O, S, CH=N, S(O) ２ , C ２ -C １０ Al Kenylene group, C ２ -C １０ Alkynylene group, C ６ -C １０ Arylene group, C ３ -C １８ F Tetracyclylene group and C ５ -C １０ Heteroarylene groups, Optionally substituted with multiple, L １ But, C １ -C １０ Alkyl group, C ６ -C １０ Aryl group, C ５ -C １０ Heteroaryl group, C １ -C １０ Halogenated alkyl group, -OC １ -C １０ a alkyl group, -OC １ -C １０ Alkylphenyl group, -C １ -C １０ Alkyl-OH, -O C １ -C １０ Halogenated alkyl group, -SC １ -C １０ Alkyl group, -SC １ -C １０ a alkylphenyl group, -C １ -C １０ Alkyl-SH, -SC １ -C １０ Alkyl halide 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 a 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 -NHC(O) (phenyl group), -N(C １ -C １０ Al kill 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 Alki phenyl group, -C(O)C １ -C １０ Haloalkyl groups, —OC(O)C １ -C １０ Al 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 N.H. ２ , -SO ２ NH (C １ -C １０ Al alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ -C １０ alkyl group), - N.H.S.O. ２ (phenyl group) and -NHSO ２ (C １ -C １０ halogenated alkyl group) and optionally having one or more substituents selected from the group consisting of 【change】 represents the site at which the group is attached to the covalent moiety; M １ The siRNA conjugate of claim 35 , wherein represents a targeting group.

43. Each L １ is independently selected from the group consisting of groups A1-A26, and any combination thereof; 【Chemistry 13】 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 14】 Each Rb is independently C １ -C １０ is an alkyl group, 【change】 The siRNA conjugate of claim 42, wherein represents the site at which the group is attached to the covalent linking moiety.

44. L １ groups A1, A4, A5, A6, A8, A10, A11, A13 and their bonds The siRNA conjugate of claim 43, wherein the siRNA conjugate is selected from the group consisting of:

45. L １ A combination of at least two bonds of groups A1, A4, A8, A10 and A11 The siRNA conjugate of claim 44 .

46. L １ is a combination of at least two bonds of groups A1, A8 and A10.

46. ​​The siRNA complex according to item 45.

47. L １ The si according to any one of claims 42 to 46, wherein the length of RNA complexes.

48. L １ The siRNA conjugate of claim 47, wherein the length of

49. j1 is an integer from 2 to 10, j2 is an integer from 2 to 10, and R′ is C １ -C ４ Al R a is one of A27, A28, A29, A30 and A31; R b But, C １ -C ５ The siRNA according to any one of claims 43 to 48, which is an alkyl group. Complex.

50. j1 is an integer of 3 to 5, j2 is an integer of 3 to 5, and R' is a methyl group or an ethyl group. and an isopropyl group, Ra is A27 or A28, and Rb is a methyl group.

50. The siRNA of claim 49, wherein the aryl group is one of an ethyl group, an isopropyl group, and a butyl group. Complex.

51. n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2 to 3. The siRNA complex according to any one of claims 42 to 50.

52. Any of claims 42 to 51, wherein each of m1, m2 and m3 is independently an integer of 2 to 5. The siRNA complex according to any one of claims 1 to 4.

53. 53. The siRNA complex according to any one of claims 42 to 52, wherein m1 = m2 = m3. 。

54. Each of said targeting groups independently binds to an asialoglycoprotein receptor on the surface of mammalian hepatocytes. The siRNA complex according to any one of claims 35 to 53, wherein the siRNA complex is a ligand having

55. 55. The method of claim 54, wherein each said targeting group is independently an asialoglycoprotein or a sugar. RNA complexes.

56. Each of said targeting groups 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 Nose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N- Trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyl N-isobutyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R -1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy- 2-Methylamino-L-glucopyranose, 4,6-dideoxy-4-formamide-2 , 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D- Glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose Nose, methyl 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-glucoheptopyrano Cid, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thio ribose, L-ribose, L-4-thioribose, 56. The siRNA complex of claim 55.

57. At least one or each of the targeting groups is galactose or N-acetylgalactosamine The siRNA complex of claim 56, wherein

58. R １０ , R １１ , R １２ , R １３ , R １４ and R １５ are independently H, a methyl group or an ethyl group. The siRNA conjugate of any one of claims 42 to 57, wherein the aryl group is a methyl group.

59. R ２ The nitrogen-containing backbone is bonded to the N atom and R ３ is bonded to a P atom in The siRNA conjugate of any one of claims 42 to 58, comprising both the nucleotide and nucleotide sites.

60. R ２ The site bonded to N on the nitrogen-containing backbone of ３ The moiety of claim 59, which is bonded to the P atom in The siRNA complexes shown above.

61. R ２ is selected from B5, B6, B5' or B6'; 【Chemistry 15】 however, 【change】 represents the site at which the group is covalently attached; q ２ is an integer of 1 to 10, 61. The siRNA complex of any one of claims 60.

62. q ２ The siRNA conjugate of claim 61, wherein is an integer from 1 to 5.

63. The siRNA complex is represented by the formula (403), (404), (405), (406), or (4 ０７）、（４０８）、（４０９）、（４１０）、（４１１）、（４１２）、（４１３）、 （４１４）、（４１５）、（４１６）、（４１７）、（４１８）、（４１９）、（４２０ ), (421) or (422) according to any one of claims 35, 42 to 62, The siRNA complex according to any one of claims 1 to 4. 【Chemistry 16】 【change】 【change】 【change】 【change】 【change】 【change】

64. The P atom in formula A59 is attached to the end of the sense strand or antisense strand of the siRNA. The end refers to the four nucleic acids preceding one end of the sense strand or the antisense strand. The siRNA conjugate of any one of claims 42 to 63, which refers to a nucleotide.

65. The P atom in formula A59 is bonded to the end of the sense strand or antisense strand of the siRNA. The siRNA conjugate of claim 64 .

66. The P atom in formula A59 is attached to the 3' end of the sense strand of the siRNA.

66. The siRNA complex according to claim 65.

67. The P atom in formula A59 is linked to the nucleic acid in the siRNA by a phosphodiester bond.

67. The method according to claim 42, wherein the 2', 3' or 5' position of the leucine is linked to the The siRNA conjugates described herein.

68. The siRNA according to any one of claims 1 to 27, or any one of claims 28 to 34. and / or the siRNA complex according to any one of claims 35 to 67. Drugs for treating and / or preventing uric acid metabolism disorders or diseases or physiological conditions caused by them Use in the preparation of.

69. A method for treating and / or preventing uric acid metabolism disorders or diseases or physiological conditions caused thereby, comprising: The siRNA according to any one of claims 1 to 27, the siRNA according to any one of claims 28 to 34 The pharmaceutical composition and / or the siRNA complex according to any one of claims 35 to 67. A method comprising administering an effective amount to a subject suffering from a uric acid metabolism disorder.

70. 69. The method according to claim 69, wherein the disease or physiological condition caused by abnormal uric acid metabolism is hyperuricemia or gout. The method according to

71. A method for suppressing expression of the XO gene in a hepatocyte, comprising the steps of: The siRNA according to any one of claims 28 to 34, the pharmaceutical composition according to any one of claims 28 to 34 and / or the pharmaceutical composition according to claim 34. Contacting the cell with an effective amount of the siRNA complex according to any one of claims 35 to 67. The method of claim 1,

72. The siRNA according to any one of claims 1 to 27, or any one of claims 28 to 34. and / or the siRNA complex according to any one of claims 35 to 67. Includes body, kit.

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