Nucleic acid, medicine composition, complex and preparation method and use of them

Abstract

To provide a siRNA suppressing expression of a complement protein 5 (C5) gene, a medicine composition including siRNA and a complex.SOLUTION: Each nucleotide of a siRNA is independently a modified or non-modified nucleotide, the siRNA includes: a sense strand; and an antisense strand. The sense strand includes a nucleotide sequence I. The nucleotide sequence I and a nucleotide sequence indicated in a sequence number 1 (5'-CUUCAUUCAUACAGACAAZ1-3') have an equal length and have 3 or less of nucleotide differences. The antisense strand includes a nucleotide sequence II, and the nucleotide sequence II and a nucleotide sequence indicated in a sequence number 2 (5'-Z2UUGUCUGUAUGAAUGAAG-3') have an equal length and have 3 or less of nucleotide differences. There is also provided a method for using the siRNA, a medicine composition and a complex of the same, for treatment and / or prevention of a disease related to a complement medium, for example myasthenia gravis (MG).SELECTED DRAWING: Figure 1C

Description

[Technical field]

[0001] The present disclosure relates to nucleic acids, pharmaceutical compositions and methods for inhibiting complement protein 5 (C5) gene expression. The present disclosure further relates to the nucleic acid, drug composition and siRNA complex. This invention relates to the preparation and use of: [Background technology]

[0002] Myasthenia gravis (MG) is primarily caused by acetylcholine receptor antibodies (AchR-Ab). It is an acquired autoimmune disease that is mediated by the neuromuscular junction, depends on cell-mediated immunity, and involves complement. This is a disease that affects the acetylcholine receptors (AChRs) on the postsynaptic membrane at the junction.

[0003] Complement protein (C5) is one of the key target points for treating myasthenia gravis. AchR-Ab binds to AchR through the involvement of complement, and AchR mediates complement-mediated cell membrane It is destroyed in large quantities by its lytic action, causing impairment of acetylcholine transmission in the postsynaptic membrane. Studies have shown that the drug specifically binds to the complement protein C5, By combining with C5α, it inhibits the cleavage of C5 into C5a and C5b and also inhibits the formation of the membrane attack complex. Inhibits the destruction of the membrane attack complex at the neuromuscular junction and the subsequent production of pro-inflammatory factors It has been shown that it blocks the immune system and exerts an immunosuppressive effect to treat myasthenia gravis.

[0004] Small interfering RNA (siRNA) Based on the mechanism of RNA interference (RNAi) The present invention provides a method for the treatment of diseases by suppressing or blocking the expression of a target gene of interest in a sequence-specific manner. It suppresses C5 gene expression at the mRNA level and inhibits the production of complement proteins. By blocking this, it maintains the body's normal immune function and suppresses the occurrence of abnormal immune responses. If it were possible to realize this, it would undoubtedly be the most ideal treatment option.

[0005] In the development of siRNA drugs to suppress C5 gene expression and treat myasthenia gravis, Appropriate siRNA and its modifications as well as effective delivery systems are key technologies. Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors of the present disclosure have demonstrated that the following siRNA complexes provided by the present disclosure: Specifically inhibits C5 gene expression, specifically targets the liver, and inhibits C5 gene expression in the liver. It has been unexpectedly discovered that the expression of the gene can be suppressed to realize treatment or prevention of myasthenia gravis. The inventors further invented highly active siRNA and pharmaceutical compositions. [Means for solving the problem]

[0007] In some embodiments, the present disclosure provides an siR having a structure shown in formula (308): Provide the NA complex.

[0008] [ka] In the formula, n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4. m1, m2, or m3 is independently an integer selected from 2 to 10; R 10 , R 11 , R 12 , R 13 , R 14 or R 15are each independently H or C 1-C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 Alkoxy is selected from the group consisting of R3 is a group having the structure shown in formula A59.

[0009] [ka] In the formula, E1 is OH, SH or BH2; Nu is an siRNA, the siRNA comprising a sense strand and an antisense strand, Each nucleotide of an iRNA is independently a modified or unmodified nucleotide. The sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reversed. The nucleotide sequence I and the nucleotide sequence II are complementary to each other to form a double-stranded region. It is one selected from the following i) to vi).

[0010] 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'-CUUCAUUCAUACAGACAAZ1-3' (SEQ ID NO: 1), 5'-Z2UUGUCUGUAUGAAUGAAG-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.

[0011] 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'-CUACAGUUUAGAAGAUUUZ5-3' (SEQ ID NO:61), 5'-Z6AAAUCUUCUAAACUGUAG-3' (SEQ ID NO:62) wherein Z5 is A and Z6 is U; 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.

[0012] 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'-GGAAGGUUACCGAGCAAUZ9-3' (SEQ ID NO: 121), 5'-Z 10 AUUGCUCGGUAACCUUCC-3' (SEQ ID NO: 122) However, Z9 is A and Z 10 is U, 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.

[0013] 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'-AGAACAGACAGCAGAAUUZ 13 -3' (SEQ ID NO: 181), 5'-Z 14 AAUUCUGCUGUCUGUUCU-3' (SEQ ID NO: 182) However, Z 13 is A and Z 14 is U, 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.

[0014] 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'-CCAAGAAGAACGCUGCAAZ 17 -3' (SEQ ID NO: 241), 5'-Z 18 UUGCAGCGUUCUUCUUGG-3' (SEQ ID NO: 242) However, Z17 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.

[0015] 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'-CCAGUAAGCAAGCCAGAAZ 21 -3' (SEQ ID NO: 301), 5'-Z 22 UUCUGGCUUGCUUACUGG-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.

[0016] 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-C10 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-C 10 Alkyl group ), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10Alkyl group)C (O)(C1-C 10 alkyl group), -N(C1-C 10 Alkyl group)C(O)(phenyl ), -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, - SO2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1-C 10 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 (halogenated alkyl group) may optionally have one or more substituents, Each L1 is independently a straight chain alkylene group of 1 to 70 carbon atoms in length, has multiple carbon atoms, C(O), NH, O, S, CH=N, S(O)2, C2-C 10 a Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 Any one selected from the group consisting of heteroarylene groups. L1 is optionally substituted with one or more of C1-C 10 Alkyl groups, C6-C 10 Ants group, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl group, -OC1 -C 10 Alkyl group, -OC1-C10 Alkylphenyl group, -C1-C 10 Alkyl- OH, -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1 -C 10 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloge alkyl groups, halogen substituents, -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 Alkylpheny ), -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)(C 1-C 10 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -N HC(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 -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylphenyl group, -C(O)C1-C 10 Haloalkyl groups, -OC(O)C1- C 10 Alkyl group, -SO2(C1-C 10alkyl group), -SO2 (phenyl group), - SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1- C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 Alki -NHSO2(phenyl group) and -NHSO2(C1-C 10 Alkyl halide may optionally have one or more substituents selected from the group consisting of JPEG2025041739000004.jpg10103 represents the site to which the group is covalently attached and M1 represents the targeting group.

[0017] In some embodiments, the present disclosure provides siRNAs capable of suppressing C5 gene expression. The siRNA comprises a sense strand and an antisense strand, and the sense strand and the antisense strand are Each nucleotide in the sense strand can be independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide. modified nucleotides, the sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence I. The strand comprises nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II means that the fluoro-modified nucleotide forms a double-stranded region in a reverse-complementary manner at least in part; are located in nucleotide sequence I and nucleotide sequence II, and are arranged from the 5' end to the 3' end. In the sense strand, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are is a fluoro-modified nucleotide, and the nucleotides at the remaining positions in the sense strand are A non-fluoro-modified nucleotide is provided from the 5' end to the 3' end of the antisense oligonucleotide. In the base strand, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are and the nucleotides at the remaining positions in the antisense strand are fluoro-modified nucleotides. is a non-fluoro modified nucleotide, and said nucleotide sequence I and said nucleotide sequence II is one selected from the above i) to vi).

[0018] In some embodiments, the present disclosure provides a method for the preparation of a siRNA comprising administering to a subject a medicament comprising administering to said subject a siRNA or a medicament comprising administering to said subject a medicament a medicament. The present invention provides a pharmaceutical composition comprising a pharmaceutical carrier.

[0019] 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.

[0020] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or siRNA complexes for the preparation of a medicament for the treatment and / or prevention of myasthenia gravis The present invention provides for use in

[0021] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or administering an effective amount of the siRNA complex to a subject suffering from myasthenia gravis. The present invention provides a method for treating and / or preventing myasthenia gravis, comprising administering to said patient a therapeutically effective amount of

[0022] 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 inhibiting the expression of the C5 gene in a mammal.

[0023] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or a kit comprising the siRNA complex. Effect of the Invention

[0024] The siRNA, drug composition and siRNA complex provided by the present disclosure have excellent stability. and / or Myasthenia gravis symptoms can be significantly treated or alleviated.

[0025] In some embodiments, the siRNA, drug composition or s The iRNA complexes show excellent target mRNA suppression activity in in vitro cell experiments. In the form, the siRNA, drug composition, or siRNA complex provided by the present disclosure , at least 20%, 30%, 40%, 50%, 60%, 70%, 80% in hepatocytes , 90% or 95% target mRNA inhibition rates are shown.

[0026] In some embodiments, the siRNA provided by the present disclosure is It showed high inhibitory activity in the IC 50 1.494~9.68 In some embodiments, the fluorescently labeled siRNA complex of the present disclosure is 8 nM. We subcutaneously injected the compound into C57 mice and performed real-time fluorescence imaging of the mice to detect the expression of the compound in organs. The distribution of fluorescence was observed, and the mice were killed 48 hours later and their organs were dissected. It was found that the A complex was mostly concentrated in the liver, and the siRNA complex provided by the present disclosure It was shown that the combination can effectively deliver siRNA specifically to the liver, These results suggest that it is possible to specifically suppress the expression of target mRNA in

[0027] In some embodiments, the siRNA, drug composition or s iRNA complexes have greater stability and / or activity in the body. In an embodiment, the present disclosure provides a siRNA, a drug composition, or a siRNA complex. is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% In some embodiments, the present disclosure provides a method for inhibiting expression of a target mRNA by 95% or 96%. The siRNA, drug composition or siRNA complex provided by %, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% C5 mR In some embodiments, the siRNP provided by the present disclosure is A. The drug composition or siRNA complex is capable of inhibiting at least 20%, 30%, 40%, 50%, or 60% of the drug composition or siRNA complex in the body. The inhibition rate of intrahepatic C5 mRNA expression was 0%, 60%, 70%, 80%, 90% or 95%. In some embodiments, the siRNA, drug composition or The siRNA complexes are expressed in the body at least 20%, 30%, 40%, 50%, 60%, 70%, The results showed that the expression of C5 mRNA in the liver was suppressed by 80%, 90%, or 95% in the animal model. In some embodiments, the siRNA, drug composition or The siRNA complexes are expressed in the body at least 20%, 30%, 40%, 50%, 60%, 70%, The results showed that the expression of C5 mRNA in the liver was suppressed by 80%, 90%, or 95% in human subjects. vinegar.

[0028] In some embodiments, the siRNA, drug composition or s The iRNA complexes did not show significant off-target effects. For example, the inhibition may be of the normal expression of a non-target gene. Binding / inhibition of on-target genes is 50%, 40%, 30%, 20% or less than the on-target gene effect. If it is 10% lower, the off-target effect is considered to be insignificant.

[0029] Thus, the siRNAs, drug compositions and siRNA complexes provided by the present disclosure and inhibiting the expression of C5 mRNA, thereby effectively treating and / or preventing myasthenia gravis. This has bright prospects for application.

[0030] 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]

[0031] [Figure 1A-1H] In order, the dose-effect curves were fitted according to the relative expression levels of C5 mRNA in HepG2 cells after transfection with different complexes 1 to 8. [Figure 2A] Fluorescence imaging images of various organs in the body of C57 mice 48 hours after administration of 5 ml / kg of 1×PBS, 3 mg / kg of Cy5-siRNA1, or 3 mg / kg of Cy5-complex 1. [Figure 2B] Fluorescence imaging images of various organs in the body of C57 mice 48 hours after administration of 5 ml / kg of 1×PBS, 3 mg / kg of Cy5-siRNA2, or 3 mg / kg of Cy5-complex 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] 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 it is not intended to be limiting in scope.

[0033] In this disclosure, C5 mRNA is set forth in Genbank accession number M57729.1. Furthermore, unless otherwise specified, the term "mRNA" as used in this disclosure refers to an mRNA having a sequence that corresponds to the The term "target gene" as used herein refers to a gene capable of transcribing the C5 mRNA, and the term "Target mRNA" refers to the C5 mRNA described above.

[0034] <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.

[0035] 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 It can substitute for nucleotides in nucleic acids, but 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.

[0036] 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.

[0037] 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...

[0038] 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.

[0039] In the present context, the siRNAs, compositions comprising siRNAs or siRNA complexes of the present disclosure are particularly In describing the preparation method of the compound, unless otherwise specified, the nucleoside monomer (nuc The leoside monomer is used in the siRNA or siRNA complex to be prepared. The modifications or modifications used in phosphoramidite solid-phase synthesis depend on the type and order of nucleotides used. represents unmodified nucleoside phosphoramidite monomers. modified RNA phosphoramidites. RNA phospho ramidites are called nucleoside phosphoramidites Phosphoramidite solid-phase synthesis is a method used to synthesize RNA known to those skilled in the art. The nucleoside monomers used in this disclosure are all commercially available. Available for purchase.

[0040] In the context of this disclosure, unless otherwise specified, "composite" means that each has a specific function. refers to a covalent bond between two or more chemical moieties that are bonded to one another, and accordingly, A "complex" is a compound formed by covalently bonding each of the chemical moieties. Furthermore, a "siRNA complex" refers to a complex that contains one or more chemical moieties with a specific function. In the following text, the term "s" refers to a compound formed by covalently binding to an iRNA. The iRNA complex may also be referred to simply as a "complex." The siRNA complex may be A siRNA complex is a general term for multiple siRNA complexes, or a siRNA complex represented by a certain chemical formula. In the context of the present disclosure, a "composite molecule" refers to a molecule that is capable of reacting with an siRNA. and ultimately form the siRNA complex of the present disclosure. It should be understood that there is.

[0041] 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.

[0042] As used herein, "alkyl" refers to both straight and branched chains having the specified number of carbon atoms. The specific number usually refers to 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

[0043] 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.

[0044] 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.

[0045] 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 .

[0046] 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 atoms and 6 to 18 carbon atoms. At least one of the rings in the ring system is fully unsaturated, i.e., it follows the Hückel theory Contains a cyclic, delocalized (4n+2) π-electron system. Aryl includes phenyl, fluorene, etc. Arylene is a substituted or unsubstituted aryl group. A aryl group is a subset of aryl and refers to a residue that is the same as aryl but has two points of attachment.

[0047] 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. .

[0048] 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.

[0049] 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).

[0050] "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, quinazolinyl, quinoxalinyl, quinolyl, tetra Hydroquinolyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydroquinazolinyl Hydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl 5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7, 8-Tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, thiazolyl Triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno [3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl -c]pridinyl) and thiophenyl / thienyl Including, but not limited to:

[0051] A variety of hydroxy protecting groups may be used in the present disclosure. Generally, the protecting groups are In this way, chemical functional groups can be rendered insensitive to certain reaction conditions while leaving the remainder of the molecule substantially can be added to and removed from the relevant functional groups in a molecule without damaging the 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).

[0052] 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.

[0053] 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.

[0054] 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 genetic alterations may have been performed, the siRNA complex or composition may be used to target a particular disease. Administered to subjects at risk for, or who have reported one or more pathological symptoms of, the disease. can be administered.

[0055] <sirna> In one aspect, the present disclosure provides six siRNAs capable of suppressing C5 gene expression.

[0056] 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.

[0057] The siRNA of the present disclosure comprises a sense strand and an antisense strand. the sense strands are the same or different in length, and the length of the sense strand is 19 to 23 nucleotides; The length of the antisense strand is 19 to 26 nucleotides. The length ratio of the sense strand to the antisense strand of the siRNA to be used was 19 / 19, 19 / 20, or 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 2 0, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / In some embodiments, the sense strand and the anti-sense strand of the siRNA may be The length ratio of the sense strands is 19 / 21, 21 / 23 or 23 / 25.

[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'-CUUCAUUCAUACAGACAAZ1-3' (SEQ ID NO: 1), 5'-Z2UUGUCUGUAUGAAUGAAG-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 These siRNAs have high ability to suppress target mRNA by siRNA, and these siRNAs are also included in the scope of the present disclosure. It is within the protection range.

[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'-CUUCAUUCAUACAGACAAZ3-3' (SEQ ID NO: 3), 5'-Z4UUGUCUGUAUGAAUGAAG-3' (SEQ ID NO: 4) wherein Z4 is the first nucleotide at the 5' end of the antisense strand, and Z3 is is selected from A, U, G or C; Z4 is a nucleotide complementary to Z3; In some embodiments, Z3 is A and Z4 is U.

[0066] 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. The nucleotide sequence III is completely reverse complementary to the nucleotide sequence I. 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 nucleotide sequence The sequence is substantially reverse-complementary or completely reverse-complementary to the second nucleotide sequence means a nucleotide sequence adjacent to the 5' end of the nucleotide sequence shown in SEQ ID NO:1 in the target mRNA. , refers to a nucleotide sequence equal in length to nucleotide sequence IV above.

[0067] 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 CU, and the base sequence of nucleotide sequence IV is AG, 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 sequence III is UCU and the base sequence of nucleotide sequence IV is AGA; 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 UUCU, and the base sequence of nucleotide sequence IV is The base sequence is AGAA, 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 CU, and the base sequence of nucleotide sequence IV is AG; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0068] 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.

[0069] <Second siRNA> According to the present disclosure, the siRNA may be a second siRNA.

[0070] 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'-CUACAGUUUAGAAGAUUUZ5-3' (SEQ ID NO:61), 5'-Z6AAAUCUUCUAAACUGUAG-3' (SEQ ID NO:62) wherein Z5 is A and Z6 is U; 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.

[0071] In some embodiments, the sense strand comprises only nucleotide sequence I, The antisense strand contains only nucleotide sequence II.

[0072] 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.

[0073] 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 A, C or G. In some embodiments, the nucleotide difference is selected from Z In some embodiments, the difference is at position Z8, where Z8 is selected from A, C, or G. , Z7 is the nucleotide complementary to Z8. A has a high ability to suppress target mRNA by siRNA, and these siRNAs are also is within the protection range.

[0074] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , essentially reverse complementary, substantially reverse complementary or completely reverse complementary.

[0075] 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'-CUACAGUUUAGAAGAUUUZ7-3' (SEQ ID NO: 63), 5'-Z8AAAUCUUCUAAACUGUAG-3' (SEQ ID NO:64) wherein Z8 is the first nucleotide at the 5' end of the antisense strand, and Z7 is is selected from A, U, G or C; Z8 is a nucleotide complementary to Z7; In some embodiments, Z7 is A and Z8 is U.

[0076] 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. The nucleotide sequence III is completely reverse complementary to the nucleotide sequence I. 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 The second nucleotide sequence is a completely reverse-complementary nucleotide sequence of the target mRNA. 61, 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.

[0077] 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 The base sequence of nucleotide sequence IV is UA, and the base sequence of nucleotide sequence IV is UA. 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 AUA and the base sequence of nucleotide sequence IV is UAU; 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 CAUA, and the base sequence of nucleotide sequence IV is The base sequence is UAUG, 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 UA, and the base sequence of nucleotide sequence IV is UA; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0078] 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.

[0079] <Third siRNA> According to the present disclosure, the siRNA may be a third siRNA.

[0080] 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'-GGAAGGUUACCGAGCAAUZ9-3' (SEQ ID NO: 121), 5'-Z 10 AUUGCUCGGUAACCUUCC-3' (SEQ ID NO: 122) However, Z9 is A and Z 10 is U, 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.

[0081] In some embodiments, the sense strand comprises only nucleotide sequence I, The antisense strand contains only nucleotide sequence II.

[0082] 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.

[0083] 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: A, C, or G. Z 12 is the difference in position, Z 12 is selected from A, C, or G. In this state, Z 11 is Z 12 The nucleotide difference is the complementary nucleotide to the nucleotide The siRNAs with these siRNAs have high ability to suppress target mRNA. A is also within the scope of protection of this disclosure.

[0084] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , essentially reverse complementary, substantially reverse complementary or completely reverse complementary.

[0085] 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'-GGAAGGUUACCGAGCAAUZ 11 -3' (SEQ ID NO: 123), 5'-Z 12 AUUGCUCGGUAACCUUCC-3' (SEQ ID NO: 124) However, the above Z 12 is the first nucleotide at the 5' end of the antisense strand, and Z 11 is selected from A, U, G or C; Z 12 is Z 11 is a nucleotide complementary to In some embodiments, Z 11 is A and Z 12 is U.

[0086] 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. The nucleotide sequence III is completely reverse complementary to the nucleotide sequence I. 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 The second nucleotide sequence is a completely reverse-complementary nucleotide sequence of the target mRNA. 121, the length of which is adjacent to the 5' end of the nucleotide sequence shown in Refers to the nucleotide sequence equivalent to column IV.

[0087] 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 G, The base of nucleotide sequence IV is C, 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 AG, and the base sequence of nucleotide sequence IV is CU, 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 CAG and the base sequence of nucleotide sequence IV is CUG; 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 CCAG, and the base sequence of nucleotide sequence IV is The base sequence is CUGG, 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 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.

[0088] 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.

[0089] <The fourth siRNA> According to the present disclosure, the siRNA may be a fourth siRNA.

[0090] 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'-AGAACAGACAGCAGAAUUZ 13 -3' (SEQ ID NO: 181), 5'-Z 14 AAUUCUGCUGUCUGUUCU-3' (SEQ ID NO: 182) However, Z 13 is A and Z 14 is U, 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.

[0091] In some embodiments, the sense strand comprises only nucleotide sequence I, The antisense strand contains only nucleotide sequence II.

[0092] 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.

[0093] 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 Z 16 but In some embodiments, the nucleotide difference is selected from: A, C, or G. Z 16 is the difference in position, Z 16 is selected from A, C, or G. In this state, Z 15 is Z 16 The nucleotide difference is the complementary nucleotide to the nucleotide The siRNAs with these siRNAs have high ability to suppress target mRNA. A is also within the scope of protection of this disclosure.

[0094] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , essentially reverse complementary, substantially reverse complementary or completely reverse complementary.

[0095] 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'-AGAACAGACAGCAGAAUUZ 15 -3' (SEQ ID NO: 183), 5'-Z 16 AAUUCUGCUGUCUGUUCU-3' (SEQ ID NO: 184) However, the above Z 16 is the first nucleotide at the 5' end of the antisense strand, and Z 15 is selected from A, U, G or C; Z 16 is Z 15 is a nucleotide complementary to In some embodiments, Z 15 is A and Z 16 is U.

[0096] 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. The nucleotide sequence III is completely reverse complementary to the nucleotide sequence I. 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 The second nucleotide sequence is a completely reverse-complementary nucleotide sequence of the target mRNA. 181, the length of which is adjacent to the 5' end of the nucleotide sequence shown in Refers to the nucleotide sequence equivalent to column IV.

[0097] 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 G, The base of nucleotide sequence IV is C, 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 GG, and the base sequence of nucleotide sequence IV is CC, 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 string III is AGG and the base sequence of nucleotide sequence IV is CCU; 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 CAGG, and the base sequence of nucleotide sequence IV is The base sequence is CCUG, 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 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.

[0098] 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.

[0099] <The fifth siRNA> According to the present disclosure, the siRNA may be a fifth siRNA.

[0100] 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'-CCAAGAAGAACGCUGCAAZ 17 -3' (SEQ ID NO: 241), 5'-Z 18 UUGCAGCGUUCUUCUUGG-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.

[0101] In some embodiments, the sense strand comprises only nucleotide sequence I, The antisense strand contains only nucleotide sequence II.

[0102] 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.

[0103] 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 with these siRNAs have high ability to suppress target mRNA. A is also within the scope of protection of this disclosure.

[0104] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , essentially reverse complementary, substantially reverse complementary or completely reverse complementary.

[0105] 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'-CCAAGAAGAACGCUGCAAZ 19 -3' (SEQ ID NO: 243), 5'-Z 20 UUGCAGCGUUCUUCUUGG-3' (SEQ ID NO: 244) However, the above Z 20 is the first nucleotide at the 5' end of the antisense strand, and Z 19 is selected from A, U, G or C; Z 20 is Z 19 is a nucleotide complementary to In some embodiments, Z 19 is A and Z 20 is U.

[0106] 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. The nucleotide sequence III is completely reverse complementary to the nucleotide sequence I. 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 The second nucleotide sequence is a completely reverse-complementary nucleotide sequence of the target mRNA. 241, the length of which is adjacent to the 5' end of the nucleotide sequence shown in Refers to the nucleotide sequence equivalent to column IV.

[0107] 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 G, The base of nucleotide sequence IV is C, 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 GG, and the base sequence of nucleotide sequence IV is CC, 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 string III is AGG and the base sequence of nucleotide sequence IV is CCU; 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 CAGG, and the base sequence of nucleotide sequence IV is The base sequence is CCUG, 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 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.

[0108] 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.

[0109] <6th siRNA> According to the present disclosure, the siRNA may be a sixth siRNA.

[0110] 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'-CCAGUAAGCAAGCCAGAAZ 21 -3' (SEQ ID NO: 301), 5'-Z 22 UUCUGGCUUGCUUACUGG-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.

[0111] In some embodiments, the sense strand comprises only nucleotide sequence I, The antisense strand contains only nucleotide sequence II.

[0112] 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.

[0113] 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 with these siRNAs have high ability to suppress target mRNA. A is also within the scope of protection of this disclosure.

[0114] In some embodiments, the nucleotide sequence I and the nucleotide sequence II , essentially reverse complementary, substantially reverse complementary or completely reverse complementary.

[0115] 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'-CCAGUAAGCAAGCCAGAAZ 23 -3' (SEQ ID NO: 303), 5'-Z 24 UUCUGGCUUGCUUACUGG-3' (SEQ ID NO: 304) However, the above Z 24 is the first nucleotide at the 5' end of the antisense strand, and Z 23 is selected from A, U, G or C; Z 24 is Z 23 is a nucleotide complementary to In some embodiments, Z 23 is A and Z 24 is U.

[0116] 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. The nucleotide sequence III is completely reverse complementary to the nucleotide sequence I. 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 The second nucleotide sequence is a completely reverse-complementary nucleotide sequence of the target mRNA. 301, the length of which is adjacent to the 5' end of the nucleotide sequence shown in Refers to the nucleotide sequence equivalent to column IV.

[0117] 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 The base sequence of nucleotide sequence IV is UA, and the base sequence of nucleotide sequence IV is UA. 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 UUA and the base sequence of nucleotide sequence IV is UAA; 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 GUUA, and the base sequence of nucleotide sequence IV is The base sequence is UAAC, 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 UA, and the base sequence of nucleotide sequence IV is UA; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0118] 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.

[0119] siRNA overhang ends and modifications The following describes the nucleotide sequence V, the nucleic acid sequence, the nucleotide modifications in the siRNA, and The description of the modified sequence can be applied to any one of the first to sixth siRNAs. That is, unless otherwise specified, the following description of siRNA refers to a first siRNA, a second siRNA, siRNA, third siRNA, fourth siRNA, fifth siRNA and sixth siRNA For example, a particular siRNA should be considered a description of the NA. If not, "the siRNA further comprises a nucleotide sequence V" means "the 1 siRNA, 2 siRNA, 3 siRNA, 4 siRNA, 5 siRNA the RNA or the sixth siRNA further comprises the nucleotide sequence V."

[0120] In some embodiments, the sense and antisense strands are different lengths, The antisense strand further comprises a nucleotide sequence V, the nucleotide sequence V having a length of 1 3 nucleotides, and attached to the 3' end of the antisense strand; This constitutes the 3' overhanging end (overhanging end). The ratio of the length of the sense strand to the antisense strand of RNA was 19 / 20, 19 / 21, 19 / 22, and 2 0 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23 , 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the nucleotide sequence V is 2 nucleotides in length: This results in a ratio of the length of the sense strand to the antisense strand of the siRNA provided by the present disclosure. It may be 19 / 21, 21 / 23 or 23 / 25.

[0121] 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 higher mRNA silencing activity.

[0122] The nucleotide at the corresponding position in the target mRNA is the nucleotide at the 5' end of the third The nucleotide or nucleotide sequence adjacent to the nucleotide sequence, The nucleotide sequence is substantially or completely reverse complementary to nucleotide sequence II. nucleotide sequence II and nucleotide sequence IV. A nucleotide sequence that is substantially or completely reverse complementary to the nucleotide sequence to be It is a column.

[0123] In some embodiments, the siRNA is The sense strand comprises the nucleotide sequence shown in SEQ ID NO:5, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: It comprises the nucleotide sequence shown in SEQ ID NO:6. 5'-CUUCAUUCAUACAGACAAZ3-3' (SEQ ID NO:5), 5'-Z4UUGUCUGUAUGAAUGAAGAG-3' (SEQ ID NO: 6)

[0124] 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'-CUCUUCAUUCAUACAGACAAZ3-3' (SEQ ID NO: 7), 5'-Z4UUGUCUGUAUGAAUGAAGAGAA-3' (SEQ ID NO: 8) wherein Z4 is the first nucleotide at the 5' end of the antisense strand, and Z3 is is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3.

[0125] In some embodiments, the siRNA is fused to the second siRNA. The sense strand comprises the nucleotide sequence shown in SEQ ID NO:65, and the antisense strand comprises the sequence It comprises the nucleotide sequence shown in number 66. 5'-CUACAGUUUAGAAGAUUUZ7-3' (SEQ ID NO: 65), 5'-Z8AAAUCUUCUAAACUGUAGUA-3' (SEQ ID NO:66)

[0126] 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'-UACUACAGUUUAGAAGAUUUZ7-3' (SEQ ID NO: 67), 5'-Z8AAAUCUUCUAAACUGUAGUAUG-3' (SEQ ID NO:68) wherein Z8 is the first nucleotide at the 5' end of the antisense strand, and Z7 is is selected from A, U, G or C, and Z8 is the nucleotide complementary to Z7.

[0127] 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'-GGAAGGUUACCGAGCAAUZ 11 -3' (SEQ ID NO: 125), 5'-Z 12 AUUGCUCGGUAACCUUCCCU-3' (SEQ ID NO: 126)

[0128] 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'-AGGGAAGGUUACCGAGCAAUZ 11 -3' (SEQ ID NO: 127), 5'-Z 12 AUUGCUCGGUAACCUUCCCUGG-3' (SEQ ID NO: 128 ) However, the above Z 12 is the first nucleotide at the 5' end of the antisense strand, and Z 11 is selected from A, U, G or C; Z 12 is Z 11 is a nucleotide complementary to do.

[0129] 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'-AGAACAGACAGCAGAAUUZ 15 -3' (SEQ ID NO: 185), 5'-Z 16 AAUUCUGCUGUCUGUUCUCC-3' (SEQ ID NO: 186)

[0130] 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'-GGAGAACAGACAGAAUUZ 15 -3' (SEQ ID NO: 187), 5'-Z 16 AAUUCUGCUGUCUGUUCUCCUG-3' (SEQ ID NO: 188 ) However, the above Z 16 is the first nucleotide at the 5' end of the antisense strand, and Z 15 is selected from A, U, G or C; Z 16 is Z 15 is a nucleotide complementary to do.

[0131] 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'-CCAAGAAGAACGCUGCAAZ 19 -3' (SEQ ID NO: 245), 5'-Z 20 UUGCAGCGUUCUUCUUGGCC-3' (SEQ ID NO: 246)

[0132] 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'-GGCCAAGAAGAACGCUGCAAZ 19 -3' (SEQ ID NO: 247), 5'-Z 20 UUGCAGCGUUCUUCUUGGCCUG-3' (SEQ ID NO: 248 ) However, the above Z 20 is the first nucleotide at the 5' end of the antisense strand, and Z 19 is selected from A, U, G or C; Z 20 is Z 19 is a nucleotide complementary to do.

[0133] 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'-CCAGUAAGCAAGCCAGAAZ 23 -3' (SEQ ID NO: 305), 5'-Z 24 UUCUGGCUUGCUUACUGGUA-3' (SEQ ID NO: 306)

[0134] 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'-UACCAGUAAGCAAGCCAGAAZ 23 -3' (SEQ ID NO: 307), 5'-Z 24 UUCUGGCUUGCUUACUGGUAAC-3' (SEQ ID NO: 308 ) However, the above Z 24 is the first nucleotide at the 5' end of the antisense strand, and Z 23 is selected from A, U, G or C; Z 24 is Z 23 is a nucleotide complementary to do.

[0135] In some embodiments, the siRNA described in this disclosure is selected from the group consisting of siRNAs described in Tables 1a-1f. siC5a1, siC5a2, siC5b1, siC5b2, siC5c1, siC5c2, siC5d1, siC5d2, siC5e1, siC5e2, siC5f 1 or siC5f2.

[0136] As discussed above, each nucleotide in the siRNA of the present disclosure may be independently modified or is an unmodified nucleotide. In some embodiments, in the siRNA of the present disclosure, In some embodiments, the nucleotides in the present disclosure are unmodified nucleotides. Some or all of the nucleotides in the siRNA are modified nucleotides. These modifications on the oxido group allow the siRNA of the present disclosure to suppress C5 gene expression. does not appreciably weaken or destroy the

[0137] In some embodiments, the siRNA of the present disclosure comprises at least one modified nucleotide. In the context of this disclosure, 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 analogue or a nucleotide having a modified base. The siRNA clearly weakened or abolished the gene expression suppression function of siRNA. For example, JK Watts, GF Deleavey, and MJ Damha,Chemically modified siRNA: to ols and applications. Drug Discov Today, 2008,13(19-20): 842-55. This is also fine.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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, The nucleotides at positions 7, 8, and 9 are fluoro-modified nucleotides, 7 or less fluoro-modified nucleotides in string II; The nucleotides at positions 2, 6, 14, and 16 of the amino acid sequence are fluoro-modified nucleotides.

[0143] 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.

[0144] In the context of this disclosure, a "fluoro-modified nucleotide" refers to a nucleotide that has a ribose residue that is The hydroxy group at the 2'-position of the group is substituted with fluorine, and the structure is represented by the following formula (7): A "non-fluoro modified nucleotide" refers to a nucleotide in which the ribose moiety of the nucleotide A nucleotide in which the 2'-hydroxy group of the nucleotide is replaced with a non-fluorine group, or a nucleotide In some embodiments, each non-fluoro modified nucleotide is a nucleoside. A nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced with a non-fluorine group It is one independently selected from a nucleotide analog.

[0145] 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.

[0146] In some embodiments, the 2'-alkoxy modified nucleotide is shown in formula (8): In some embodiments, the nucleotide sequence is a 2'-methoxy (2'-OMe) modified nucleotide. In the present invention, the 2'-substituted alkoxy modified nucleotide is, for example, a 2'-O In some embodiments, the nucleotide may be a 2'-methoxyethyl (2'-MOE) modified nucleotide. In one embodiment, the 2'-amino (2'-NH2) modified nucleotide is shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is represented by formula (11): will be done.

[0147] [ka]

[0148] 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, The nucleotide may be a nucleotide or an acyclic nucleotide.

[0149] Bridged nucleic acid (BNA) is a binding BNA refers to a nucleotide that is restricted or inaccessible to the nucleotide sequence. It may also contain a bridged structure with a "fixed" C3'-endo sugar puckering. The bridge is introduced at the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, the BNA is an LNA as shown in formula (12), 3), cET BNA shown in formula (14), or the like.

[0150] [ka]

[0151] 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).

[0152] [ka]

[0153] In the above formula (15) and formula (16), R is H, OH or alkoxy (O-alkyl). The signal is selected from the following:

[0154] 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.

[0155] [ka]

[0156] 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.

[0157] 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.

[0158] 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

[0159] 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.

[0160] 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. from the 5' end to the 3' end in the antisense strand of the siRNA Nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluoro-modified. The remaining nucleotides in the antisense strand of the siRNA are is an oxy-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. , 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 remaining nucleotides in 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 remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides. It is.

[0161] In some embodiments, the siRNA provided by the present disclosure comprises a siRNA selected from the group consisting of siRNAs listed in Tables 1a-1f. siC5a1-M1, siC5a1-M2, siC5a1-M3, and siC 5a2-M1, siC5a2-M2, siC5a2-M3, siC5b1-M1, siC 5b1-M2, siC5b1-M3, siC5b2-M1, siC5b2-M2, siC 5b2-M3, siC5c1-M1, siC5c1-M2, siC5c1-M3, siC 5c2-M1, siC5c2-M2, siC5c2-M3, siC5d1-M1, siC 5d1-M2, siC5d1-M3, siC5d2-M1, siC5d2-M2, siC 5d2-M3, siC5e1-M1, siC5e1-M2, siC5e1-M3, siC 5e2-M1, siC5e2-M2, siC5e2-M3, siC5f1-M1, siC 5f1-M2, siC5f1-M3, siC5f2-M1, siC5f2-M2 or si It is either C5f2-M3.

[0162] The siRNAs with the above modifications are not only inexpensive 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. It has improved resistance to enzyme hydrolysis.

[0163] 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.

[0164] [ka]

[0165] These modifications stabilize the double-stranded structure of siRNA and enhance the specificity of base pairing. and maintain a high affinity.

[0166] 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.

[0167] In some embodiments, the siRNA provided by the present disclosure comprises a siRNA selected from the group consisting of siRNAs listed in Tables 1a-1f. siC5a1-M1S, siC5a1-M2S, siC5a1-M3S, siC5a2-M1S, siC5a2-M2S, siC5a2-M3S, siC5b1- M1S, siC5b1-M2S, siC5b1-M3S, siC5b2-M1S, siC 5b2-M2S, siC5b2-M3S, siC5c1-M1S, siC5c1-M2S , siC5c1-M3S, siC5c2-M1S, siC5c2-M2S, siC5c2 -M3S, siC5d1-M1S, siC5d1-M2S, siC5d1-M3S, si C5d2-M1S, siC5d2-M2S, siC5d2-M3S, siC5e1-M1 S, siC5e1-M2S, siC5e1-M3S, siC5e2-M1S, siC5e 2-M2S, siC5e2-M3S, siC5f1-M1S, siC5f1-M2S, s iC5f1-M3S, siC5f2-M1S, siC5f2-M2S or siC5f2- It is one of the M3S.

[0168] 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.

[0169] 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: .

[0170] [ka]

[0171] 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:

[0172] [ka] In the formula, R is selected from H, OH, methoxy, and fluorine; Base represents a base; A, U , C, G or T.

[0173] 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.

[0174] In some embodiments, the siRNA provided by the present disclosure comprises a siRNA selected from the group consisting of siRNAs listed in Tables 1a-1f. siC5a1-M1P1, siC5a1-M2P1, siC5a1-M3 P1、siC5a2-M1P1、siC5a2-M2P1、siC5a2-M3P1、s iC5b1-M1P1、siC5b1-M2P1、siC5b1-M3P1、siC5b 2-M1P1、siC5b2-M2P1、siC5b2-M3P1、siC5c1-M1 P1、siC5c1-M2P1、siC5c1-M3P1、siC5c2-M1P1、s iC5c2-M2P1、siC5c2-M3P1、siC5d1-M1P1、siC5d 1-M2P1、siC5d1-M3P1、siC5d2-M1P1、siC5d2-M2 P1、siC5d2-M3P1、siC5e1-M1P1、siC5e1-M2P1、s iC5e1-M3P1、siC5e2-M1P1、siC5e2-M2P1、siC5e 2-M3P1、siC5f1-M1P1、siC5f1-M2P1、siC5f1-M3 P1、siC5f2-M1P1、siC5f2-M2P1、siC5f2-M3P1、s iC5a1-M1SP1、siC5a1-M2SP1、siC5a1-M3SP1、si C5a2-M1SP1、siC5a2-M2SP1、siC5a2-M3SP1、siC 5b1-M1SP1、siC5b1-M2SP1、siC5b1-M3SP1、siC5 b2-M1SP1、siC5b2-M2SP1、siC5b2-M3SP1、siC5c 1-M1SP1、siC5c1-M2SP1、siC5c1-M3SP1、siC5c2 -M1SP1、siC5c2-M2SP1、siC5c2-M3SP1、siC5d1- M1SP1、siC5d1-M2SP1、siC5d1-M3SP1、siC5d2-M 1SP1、siC5d2-M2SP1、siC5d2-M3SP1、siC5e1-M1 SP1, siC5e1-M2SP1, siC5e1-M3SP1, siC5e2-M1S P1, siC5e2-M2SP1, siC5e2-M3SP1, siC5f1-M1SP 1, siC5f1-M2SP1, siC5f1-M3SP1, siC5f2-M1SP1 , siC5f2-M2SP1 or siC5f2-M3SP1.

[0175] The inventors of the present disclosure have demonstrated the plasma and lysosomal stability of the siRNAs provided by the present disclosure. We unexpectedly found that not only was the activity significantly improved, but that the activity of suppressing the target mRNA was also high.

[0176] 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.

[0177] <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.

[0178] 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.

[0179] 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).

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] [ka] During the ceremony, X 101 Or X 102 are each independently O, S, NA or CA, and A is hydrogen. Or C1-C 20 is a hydrocarbon chain, Y 101 or Z 101 are each independently C=O, C=S, S=O, CH-OH or S O2, R 101 , R 102 , R 103 , R 104 , R 105 , R 106 or R 107 Each each independently being hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, a cyclic or an acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic group, substituted or unsubstituted, Branched or straight chain acyl groups, substituted or unsubstituted branched or straight chain aryl groups, substituted or unsubstituted 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).

[0188] [ka] In the formula, g, e, and f each independently represent an integer from 1 to 6, and "HCC" represents a hydrocarbon chain. and each *N represents a nitrogen atom in formula (201).

[0189] 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.

[0190] 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).

[0191] [ka]

[0192] 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 * represents R 103 and nitrogen in formula (201) Each H at any * position is a bond to the nitrogen atom in formula (201). It may be substituted for binding.

[0193] The compound of formula (201) was prepared according to the method described in CN103380113A. Good too.

[0194] In some embodiments, the organic amine is an organic amine represented by formula (214): and / or an organic amine represented by formula (215).

[0195] [ka]

[0196] 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).

[0197] 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- 50), and may be, for example, (50-70):(20-40):(3-20). .

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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:(2–5).

[0204] 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.

[0205] Here, the concentration or dilution is carried out before, after, or simultaneously with the removal of impurities. As a method for removing impurities, various conventional methods may be adopted, for example, The mixture was ultrafiltered at 100KDa using a tangential flow system and a hollow fiber column. The ultrafiltration exchange solution may be phosphate buffered saline (PBS) at pH 7.4. For this purpose, various conventional methods may be used, for example, filtration through a 0.22 μm filter. It may also be disinfected by

[0206] siRNA complex The present disclosure relates to a siGe that includes the above-mentioned siRNA and a conjugated group that is conjugated and bound to the siRNA. An RNA complex is provided.

[0207] Generally, the conjugate group comprises at least one pharma- ceutically acceptable targeting group and an optional linker. the siRNA, the linker and the targeting group are sequentially bonded In some embodiments, the targeting groups are 1 to 6. In one embodiment, the number of targeting groups is 2 to 4. The siRNA molecule is It may be non-covalently or covalently conjugated, for example, covalently conjugated to the conjugating group. The conjugation site between the siRNA and the conjugation group may be at the 3' end or the 3' end of the sense strand of the siRNA. may be at the 5' end of the antisense strand, or at the 5' end of the siRNA. In some embodiments, the conjugation site between the siRNA and the conjugation group may be in a row. is at the 3' end of the sense strand of the siRNA.

[0208] 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.

[0209] 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.

[0210] 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. .

[0211] 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 residue, lipoprotein (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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] In some embodiments, when the targeting group is N-acetylgalactosamine, A suitable linker may be the structure shown in formula (301).

[0219] [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.

[0220] [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.

[0221] [ka] L C is 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

[0222] 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).

[0223] [ka] In the formula, the double helix structure represents the siRNA.

[0224] 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. This is also possible.

[0225] 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.

[0226] [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.

[0227] In some embodiments, when the targeting group is N-acetylgalactosamine, A suitable linker may be the structure shown in formula (306).

[0228] [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 . In some embodiments, when l=2, the siRNA complex has the formula (30 7) has the structure shown in

[0229] [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.

[0230] 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.

[0231] In some embodiments, the siRNA complex has the structure shown in formula (308): has.

[0232] [ka] 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, or m3 is 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 alkoxy groups; R3 is a group having the structure shown in formula A59.

[0233] [ka] wherein E1 is OH, SH, or BH2, and Nu is an siRNA of the disclosure.

[0234] R2 is a linear alkylene group of 1 to 20 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, R2 is optionally substituted with C1-C 10 Alkyl groups, C6-C 10 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(C 1-C 10 alkylphenyl group), cyano group, nitro group, -COH, -C(O)O(C 1-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- 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 nyl group), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylpheny group, -C(O)C1-C 10 Haloalkyl group, -OC(O)C1-C 10 Alkyl groups, -SO2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1-C1 0Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 (alkyl group) , -SO2NH(phenyl group), -NHSO2(C1-C 10 alkyl group), -NHSO 2 (phenyl group) and -NHSO2 (C1-C 10 halogenated alkyl groups) may optionally bear one or more substituents, Each L1 is independently a straight chain alkylene group of 1 to 70 carbon atoms in length, has multiple carbon atoms, C(O), NH, O, S, CH=N, S(O)2, C2-C 10 a Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 Heteroarylene groups, is optionally substituted with multiple, and L1 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 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 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Al halide alkyl groups, halogen substituents, -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-C1 0 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O )(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 a 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 Al alkylphenyl group, -C(O)C1-C 10 Haloalkyl group, -OC(O)C1-C 10 a alkyl group, -SO2(C1-C 10 Alkyl group), -SO2(phenyl group), -SO2( C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 a alkyl group), -SO2NH (phenyl group), -NHSO2(C1-C 10 alkyl group), -NHSO2(phenyl group) and -NHSO2(C1-C 10 halogenated alkyl group) and optionally having one or more substituents selected from the group consisting of:

[0235] In some embodiments, L1 is a group selected from (A1)-(A26) or any bond thereof. The structures and definitions of (A1) to (A26) may be selected from the group consisting of the following combinations: That is correct.

[0236] [ka] In the formula, each j1 is independently an integer from 1 to 20, and each j2 is independently an integer from 1 to 20. the law of nature, Each R' is independently C1-C 10 is an alkyl group, Each Ra is a group represented by the formulae (A27) to (A45) or any combination of bonds thereof. The compound is selected from the group consisting of:

[0237] [ka] Each Rb is independently C1-C 10 is an alkyl group, JPEG2025041739000027.jpg1083 represents the site to which the group is covalently attached.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] In some embodiments, m1, m2, or m3 each independently represents an integer of 2 to 10. When the number of M1 targeting groups is selected from the number of M1 targeting groups, the spatial position between the M1 targeting groups and the asialo The siRs provided by the present disclosure can be adapted to bind to glycoprotein receptors. To make the NA complex simpler, easier to synthesize, and / or less costly, In some embodiments, m1, m2, or m3 are each independently an integer from 2 to 5. and in some embodiments, m1=m2=m3.

[0242] 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 15 are both H.

[0243] 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.

[0244] R2 is selected to achieve a bond between the N atom on the nitrogen-containing backbone and A59. In the present context, a "nitrogen-containing backbone" is R 10 , R 11 , R 12 , R 13 , R 14 Reach BiR 15 It refers to a chain structure in which carbon atoms with carbon atoms bonded to them and N atoms are bonded to each other. and R2 is any group capable of binding the A59 group to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, the linker group may be any linker group 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, When preparing the siRNA complex of formula (308), the R2 group is preferably a nitrogen-containing backbone. The site bonded to the N atom above 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 may be The bonded site forms an amide bond with the N atom and is bonded to the P atom on R3. forms a phosphate bond with the P atom, and in some embodiments, R2 is B5 , B6, B5' or B6'.

[0245] [ka] During the ceremony, JPEG2025041739000029.jpg1497 represents the site to which the group is covalently attached.

[0246] The value of q2 may range from 1 to 10, and in some embodiments, q 2 is an integer from 1 to 5.

[0247] L1 binds the M1 targeting group to the N on the nitrogen-containing backbone, forming a siRNP 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.

[0248] 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.

[0249] 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.

[0250] 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).

[0251] [ka] JPEG2025041739000031.jpg217105JPEG2025041739000032.jpg225104JPEG2025041739000033.jpg21710 9JPEG2025041739000034.jpg250104JPEG2025041739000035.jpg233104JPEG2025041739000036.jpg17389

[0252] 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. After the siRNA is loaded, it releases the single antisense strand of the siRNA as it unwinds, and the C5 It blocks the process of mRNA translation into protein, suppressing gene expression of the complement protein C5. It is possible.

[0253] In some embodiments, P in formula A59 represents a nucleotide in the siRNA. Any possible position on the nucleotide, for example, the 5' position of a nucleotide, the 2' position of a nucleotide, In some embodiments, the nucleotide may be attached to the 3' position of the nucleotide or to the base of the nucleotide. In the formula A59, the P atom is linked to the si by forming a phosphodiester bond. It may be attached to the 2', 3' or 5' position of the nucleotide in the RNA. In one embodiment, the P atom in formula A59 is the 3'-terminal nucleotide of the sense strand of the siRNA. The 3' hydroxyl group of the thiol is dehydrogenated to the oxygen atom (at this time, The P atom can be regarded as the P atom of a phosphate group contained in the siRNA), or The P atom in formula A59 is the 2'- of one nucleotide in the sense strand of the siRNA. or is attached to the nucleotide by replacing the hydrogen in the hydroxy group, or is represented by the formula A59 The P atom in the 5' hydroxyl group of the 5' terminal nucleotide of the sense strand of the siRNA is attached to the nucleotide by replacing a hydrogen atom of

[0254] The inventors of the present disclosure have found that the siRNA complex of the present disclosure has significantly improved stability in plasma and The target effect was reduced and the C5 mRNA silencing activity was enhanced. In some embodiments, the siRNA of the present disclosure is selected from the group consisting of those listed in Tables 1a-1. f. siRNA complexes containing these siRNAs. The combination exhibits higher C5 mRNA silencing activity.

[0255] [Table 1a] [Table 1b] [Table 1c] [Table 1d] [Table 1e] [Table 1f]

[0256] 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.

[0257] 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.

[0258] <Preparation of siRNA complex represented by formula (308)> The siRNA complex represented by formula (308) may be prepared by any reasonable synthetic route. good.

[0259] 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.

[0260] 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 represented by formula (321) below is a conjugated molecule. It is also called.

[0261] [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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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').

[0266] [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, JPEG2025041739000045.jpg1497 represents the site to which the group is covalently attached.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] In some embodiments, R4 is selected from the group consisting of formula (B9), (B10), (B9′), (B1 0'), (B11), (B12), (B11') or (B12') .

[0272] [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, JPEG2025041739000047.jpg1497 represents the site to which the group is covalently attached. In some embodiments, q1 is 1 or In some embodiments, q2 is an integer from 1 to 5. In some embodiments, R4 comprises a structure shown in formula (B9) or (B10). In some embodiments, R4 comprises a structure shown in formula (B11) or (B12).

[0273] 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).

[0274] The definition of L1 is as described above.

[0275] 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 is any one of formulas (A1) to (A26) or This includes combinations of:

[0276] 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.

[0277] 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 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, 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.

[0278] In some embodiments, each S1 is independently selected from the group consisting of formulae A46-A54. is selected from the group.

[0279] [ka]

[0280] In some embodiments, S1 is of formula A49 or A50.

[0281] 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.

[0282] 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).

[0283] 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.

[0284] 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.

[0285] 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

[0286] 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.

[0287] The coupling reaction conditions and coupling reagents may be any suitable coupling reaction conditions. In some embodiments, the solid phase synthesis method employed may include the following conditions and reagents: The same conditions and reagents as those used in the coupling reaction in may be used.

[0288] 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.

[0289] 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 siRNA complex is synthesized in the direction of It is attached to the 3' end of the sense strand.

[0290] 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, conditions for capping reaction, type and dosage of capping reagent, conditions for oxidation reaction, oxidation The types and amounts of reagents, sulfurization reaction conditions, and the types and amounts of sulfurization reagents are included. Various reagents, dosages and conditions are used.

[0291] For example, in some embodiments, in steps (2) and (3), the solid phase synthesis Then, the following conditions may be used:

[0292] 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.

[0293] 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 be coupled is 1:1 to 1:100. The reaction time and the selection of coupling reagents are the same as above. be.

[0294] 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 1:1 :2~2:2:1.

[0295] The oxidation reaction conditions are a temperature of 0 to 50° C., and in some embodiments, 15 to 35° C. The reaction time is from 1 to 100 seconds, in some embodiments from 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 oxidation reagent to the nucleic acid sequence bound to the solid support in the coupling step is 1. The ratio may be from 5:1 to 100:1, and in some embodiments, from 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a 3:1 mixture of tetrahydrofuran:water:pyridine. The sulfurization reaction is carried out in a mixed solvent of 1 to 1:1:3. The sulfurization reaction conditions are temperature of 0 to 50°C, several In some embodiments, the temperature is 15 to 35° C., and the reaction time is 50 to 2000 seconds. is 100 to 1000 seconds, and the sulfurizing reagent is, in some embodiments, xanthan hydride. The molar ratio of the sulfurization reagent to the nucleic acid sequence that is bound to the solid support in the coupling step is The ratio 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 1:1.

[0296] After all nucleoside monomers have been coupled and before annealing, the method further comprises: and 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 phosphated. This includes removing the protecting groups on the base and on the ligand, purifying and desalting.

[0297] 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 form a complex shown in formula (308). Here, the concentrated ammonia water may be 25 to 30% by weight of ammonia water. The dose of concentrated ammonia water was 0.2 ml / μmol to 100 μmol per siRNA sequence of interest. It may be 0.8 ml / μmol.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] Annealing methods are also well known to those skilled in the art. The sense strand (SS strand) and the antisense strand (AS strand) were mixed in an equal molar ratio with water for injection and incubated for 70-90 min. 5°C and then cooled to room temperature, allowing the formation of a double-stranded structure through hydrogen bonds. In this manner, the siRNA complex shown in formula (308) can be obtained.

[0303] After obtaining the complex, in some embodiments, the complex may be purified by, for example, liquid chromatography. Using methods such as tandem mass spectrometry, the synthesized compound shown in formula (308) was analyzed by molecular weight detection, etc. We clarified the characteristics of the siRNA complexes synthesized and confirmed that the siRNA complexes synthesized were The siRNA complex shown in formula (308) and the sequence of the synthesized siRNA is the desired siRNA sequence, e.g., one of the sequences shown in Tables 1a-1f. It is also possible.

[0304] 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:

[0305] [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).

[0306] [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.

[0307] 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 4 The temperature is 0°C and the reaction time is 20 to 30 hours.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] The ion exchange reaction is carried out by reacting the compound of formula (321) with the desired carboxylic acid or carboxylate. The method of ion exchange is known to those skilled in the art. Using the solution and exchange conditions, M + A complex molecule having a cation can be obtained, where In some embodiments, the ion exchange reaction is carried out by triethylamine. The concentration of the triethylamine phosphate solution is 0. In some embodiments, the triethylamine phosphate is 0.2 to 0.8M. The concentration of the solution is 0.4 to 0.6 M, and the triethyl The dosage of the amine phosphate solution is 3 to 6 L / mol, and in a further embodiment 4 to 5 L / mol.

[0313] 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.

[0314] 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.

[0315] 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. .

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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 may be a capping reagent known to those skilled in the art for use in solid-phase synthesis of siRNA. may also be used.

[0320] 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.

[0321] 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.

[0322] In some embodiments, the capping reagent comprises equimolar amounts 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.

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] In some embodiments, the method for preparing a compound of Formula (321) comprises the steps of: 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.

[0328] 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.

[0329] [ka]

[0330] 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.

[0331] 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.

[0332] 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.

[0333] The conditions for the oxidation reaction are 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 oxidation reagent may be, for example, iodine. In some embodiments, the iodine may be provided as iodine water. The molar ratio of the oxidation reagent to the nucleic acid sequence bound to the solid support is 1:1 to 100:1, e.g. For example, the ratio may be 5:1 to 50:1. The reaction is carried out in a mixed solvent of dihydrofuran:water:pyridine=3:1:1 to 1:1:3.

[0334] In some embodiments, R6 is one of the groups of formula B7 or B8.

[0335] [ka] In the formula, q 2、 R k The definition is as described above.

[0336] 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.

[0337] [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.

[0338] 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 4 The reaction temperature is 0°C and the reaction time is 2 to 16 hours.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] [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.

[0347] As the compound of formula (316), for example, the compound described in J. Am. Chem. Soc. 201 Compounds disclosed in US Pat. No. 4,136,16958-16961 may be used, or compounds of the formula ( 316) The compounds can be prepared by those 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 US Pat. No. 8,106,022 B2, several formulas (3 16), the entire contents of which are incorporated herein by reference in their entirety. INCORPORATED INTO THE SPECIFICATION.

[0348] In some embodiments, the condensation reaction conditions include a reaction temperature of 0 to 100° C. The reaction time is 0.1 to 24 hours, and in some embodiments, the reaction temperature is 10 to 40° C. and the reaction time is 0.5 to 16 hours.

[0349] 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.

[0350] 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.

[0351] 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.

[0352] 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.

[0353] 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.

[0354] 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, and n3 = 2, and each R 10 , R 11 , R 12 , R 13 , R 14 , R 15 When both are H, equation (320) The compound can be purchased commercially from Alfa Aesar.

[0355] 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.

[0356] 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 siRNA complexes for the preparation of a medicament for the treatment and / or prevention of myasthenia gravis The present invention provides for use in

[0357] 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. A method for preventing and / or treating myasthenia gravis is provided.

[0358] siRNA active components of the present disclosure can be administered to a subject in need thereof to induce RNase activity. Achieving the purpose of preventing and / or treating myasthenia gravis through the mechanism of intervention Therefore, the siRNA and / or drug composition and / or siRNA of the present disclosure can be used. The A complex is used for the prevention and / or treatment of myasthenia gravis, or for the prevention and treatment of myasthenia gravis. and / or for the preparation of a medicament for treatment.

[0359] 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. Considering the possibility of providing preventive and / or therapeutic measures for diabetes, several practical In embodiments, an administration method is employed that can deliver the drug to the liver.

[0360] 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 than once every three weeks, once a month, twice a month, three months, six months or once a year.

[0361] 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), ED 50 (In a quantitative response, this refers to the dose that can induce 50% of the maximum response intensity. IC refers to the dose at which a positive sexual response occurs in 50% of experimental subjects 50 The concentration of inhibitor / drug at which the quantitative response is half-inhibited may be measured. A range of human dosages can be derived based on data obtained from nutritional analyses and animal studies. do.

[0362] Administering the siRNA, drug composition, and / or siRNA complex described in this disclosure In this case, for example, male or female C57BL / 6J mice aged 6 to 12 weeks and weighing 18 to 25 g are used. (i) for a siRNA complex, The dosage may be 0.001 to 100 mg / kg body weight, and in some embodiments, 0. In some embodiments, the dose is 0.01 to 50 mg / kg body weight, and in some embodiments, the dose is 0.05 to 20 mg / kg body weight. and in some embodiments, 0.1 to 15 mg / kg body weight. In some embodiments, the dose is 0.1 to 10 mg / kg body weight, and (ii) the siR A pharmaceutical composition formed by NA and a pharma- ceutical acceptable carrier, The dosage may be from 0.001 to 50 mg / kg body weight, and in some embodiments is 0.0 1 to 10 mg / kg body weight, and in some embodiments, 0.05 to 5 mg / kg body weight. and in some embodiments, 0.1 to 3 mg / kg body weight.

[0363] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or contacting a liver cell with an effective amount of the siRNA complex of the present disclosure. Alternatively, a drug composition and / or a siRNA complex is introduced into the hepatocytes to induce the mechanism of RNA interference. The present invention relates to a method for suppressing the expression of C5 gene in hepatocytes by a method comprising the steps of: The present invention provides a method for inhibiting the expression of the C5 gene in the hepatocytes. The hepatocytes are SMMC-7 721, HepG2, Huh7, or other hepatoma cell lines or isolated primary hepatocytes. In some embodiments, the cells are HepG2 liver cancer cells.

[0364] The method provided by the present disclosure inhibits expression of the C5 gene in cells. 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

[0365] <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.

[0366] 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.

[0367] 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.

[0368] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto. do not have. EXAMPLES

[0369] 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).

[0370] siRNA, siRNA complex or negative control against C5 gene synthesized according to the present disclosure When cells were transfected with control siRNA or siRNA complex, Lipofectamine as a transfection reagent TM 2000 (Invitrogen en) and refer to the instructions provided by the manufacturer for specific operations. .

[0371] Unless otherwise stated, all reagent proportions provided below are by volume (v / v). It is calculated.

[0372] The experimental data are

number

[0373] (Preparation Example 1) Preparation of Complex 1 In this preparation example, complex 1 (i.e., L10-siC5a1M1SP) was synthesized. The siRNA complexed to the complex consisted of a sense strand and an antisense strand corresponding to complex 1 in Table 3. It has a strand sequence.

[0374] (1-1) Synthesis of Compound L-10 Compound L-10 was synthesized according to the following method.

[0375] [ka]

[0376] (1-1-1) Synthesis of Complex Terminal Segment GAL-5 [ka]

[0377] (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.

[0378] (1-1-1b) Synthesis of GAL-3 The GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was The mixture was dissolved in 3 ml of anhydrous 1,2-dichloroethane and heated in an ice-water bath under nitrogen protection to obtain a 24.0 mL sample. g of trimethylsilyl trifluoromethanesulfonate (TMSOTf, CAS number: 27 607-77-8, purchased from Macklin, 108.0 mmol) was added and reacted at room temperature overnight. He responded.

[0379] 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.

[0380] (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 and the aqueous phase was extracted once with 300 ml of dichloroethane. 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.

[0381] (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 mixture was extracted three times with 200 ml of water per 100 ml of water, and the organic phase was discarded. The pH of the aqueous phase was adjusted to about 3 with solid citric acid. The extract was then extracted three times with dichloromethane, each time at 200 ml. The organic phases were combined and washed with anhydrous sulfuric acid. The solvent was evaporated to dryness under reduced pressure to give 6.85 g of a white foamy solid product, GAL I got -5. 1 H NMR (400 MHz,DMSO) δ 12.01 (br, 1H),7.83 (d,J = 9.2 Hz,1H),5.21 (d,J = 3 .2 Hz,1H),4.96 (dd,J = 11.2,3.2 Hz,1H),4 .49 (d,J = 8.4 Hz,1H),4.07-3.95 (m,3H),3 .92-3.85 (m,1H), 3.74-3.67 (m,1H), 3.48-3. 39 (m,1H),2.20 (t,J = 6.8 Hz,2H),2.11 (s ,3H),2.00 (s,3H),1.90 (s,3H),1.77 (s,3H) ,1.55-1.45 (m,4H).

[0382] (1-1-2) Synthesis of L-8 [ka]

[0383] 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.

[0384] (1-1-3a) Synthesis of A-1 [ka]

[0385] 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.

[0386] (1-1-3b) Synthesis of L-7 [ka]

[0387] 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.

[0388] Synthesis of (1-1-4)L-9 [ka]

[0389] 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. Wash the reaction mixture with diethylamine phosphate, and then wash the aqueous phase with dichloromethane, 3 times with 5 ml each. The organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. Normal phase silica gel of 00 to 300 mesh was used, and silica gel was purified with 1 wt% triethylamine. The acidity of the column was neutralized, and the column was equilibrated with dichloromethane and diluted with 1 wt‰ triethylamine. Gradient elution with dichloromethane:methanol = 100:18 to 100:20, product eluate The extract was collected and the solvent was evaporated to dryness under reduced pressure to obtain 31.0 g of pure L-9 complex molecule. 1 HN MR (400 MHz,DMSO) δ 8.58 (d,J = 4.2 Hz,1 H), 7.94-7.82 (m,3H), 7.41-7.29 (m,5H), 7.2 2 (d,J = 8.1 Hz,5H),6.89 (d,J = 8.3 Hz,4 H),5.49-5.37 (m,1H),5.21 (d,J = 3.0 Hz,3 H),4.97 (d,J = 11.1 Hz,3H),4.49 (d,J = 8 .2 Hz,3H),4.02 (s,9H),3.88 (dd,J = 19.4, 9.4 Hz, 3H), 3.77-3.65 (m, 9H), 3.50-3.39 (m ,6H),3.11-2.90 (m,5H),2.61-2.54 (m,4H),2 .47-2.41 (m,2H),2.26-2.17 (m,2H),2.15-1. 95 (m,22H), 1.92-1.84 (m,9H), 1.80-1.70 (m ,10H),1.65-1.35 (m,17H),1.31-1.19 (m,4H) ,0.96 (t,J = 7.1 Hz,9H). MS m / z:C 94 H 132 N 7O 38 , [M-DMTr] + , theoretical value: 1664.72, actual value: 1665.03.

[0390] (1-1-5) Synthesis of Compound L-10 [ka]

[0391] In this step, the L-9 conjugate molecule is bound to a solid support to form the L-10 compound. was prepared.

[0392] 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 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 then rinsed three times with water and dried for 18 hours using a vacuum oil pump. (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) The capping reaction was carried out by adding the above. 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.

[0393] [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.

[0394] (1-2) Synthesis of the sense strand of complex 1 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. The sense strand SS of the complex 1 in Table 3 was synthesized by combining the nucleoside monomers. Each time a monomer is bonded, four steps are performed: deprotection, coupling, capping, and oxidation or sulfurization. When the two nucleotides are linked by a phosphate ester, the following nucleotides are formed: When combining thiol side monomers, four steps are performed: deprotection, coupling, capping, and oxidation. When two nucleotides are linked by a thiophosphate, the following reaction occurs: The conjugation of nucleoside monomers involves four steps: deprotection, coupling, capping, and sulfurization. The synthesis conditions were as follows:

[0395] 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.

[0396] 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.

[0397] 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 to be 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.

[0398] The conditions for each oxidation reaction were the same: temperature 25°C, reaction time 15 seconds, and the oxidation reagent was concentrated. The iodine solution is 0.05M in concentration. The iodine and the solid support bound in the coupling step are The molar ratio of the nucleic acid sequence to be synthesized is 30:1. The reaction is tetrahydrofuran:water:pyridine= The reaction was carried out using a 3:1:1 mixed solvent.

[0399] 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.

[0400] 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.

[0401] 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.

[0402] 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.

[0403] 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 measured value was consistent with the theoretical value, and the L-9 complex molecule was found at the 3' end. It was shown that the conjugated sense strand SS was synthesized.

[0404] (1-3) Synthesis of antisense strand of complex 1 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) was used as the starting material, and the multiple The antisense strand AS of the combination 1 was synthesized. For the antisense strand, the 5'-terminal 1st Since the first nucleotide has a 5'-phosphate, it can be synthesized by the solid-phase phosphoramidite method. During the process of preparing the sense strand, the last nucleoside monomer of the antisense strand is attached. After that, four reactions, namely, deprotection, coupling, capping, and oxidation, were carried out to obtain the CPR-I model. A 5' end of the antisense strand was added to the 5' end of the antisense strand. Deprotection in solid phase synthesis method except for coupling and forming 5'-phosphate ester modification , coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions The conditions were the same as those for the synthesis of the sense strand.

[0405] [ka]

[0406] the conditions of the deprotection, coupling, capping and oxidation reactions used in said conjugation; The conditions for cleavage, deprotection, purification and desalting were the same as those for the synthesis of the sense strand. The antisense strand was isolated by ion exchange chromatography (IEX-HPLC). The purity of the antisense strand was detected and analyzed by liquid chromatography-mass spectrometry (LC-MS). The molecular weight was analyzed. As a result, the measured value was consistent with the theoretical value, and the antisense oligonucleotide had the target sequence. This shows that the AS chain was synthesized.

[0407] (1-4) Synthesis of Complex 1 The sense strand and the antisense strand were each dissolved in water for injection to obtain a 40 mg / mL solution. The mixture was mixed in an equimolar ratio, heated at 50°C for 15 min, cooled at room temperature, and then annealed. The product was obtained and freeze-dried to obtain a freeze-dried powder. Ultrapure water (Milli-Q ultrapure water system) The complex was diluted to a concentration of 0.2 mg / mL using a 100% ethanol solution with a resistivity of 18.2 MΩ*cm (25°C). After dilution to 100 ml, the mixture was analyzed by liquid chromatography-mass spectrometry (LC-MS, Liquid C Chromatography-Mass Spectrometry, Waters The molecular weight was detected using a chromatograph (purchased from Eppendorf, model number: LCT Premier). The synthesized complex 1 is a double-stranded nucleic acid sequence of a purpose-designed type containing the L-9 complex molecule. Its structure is shown in formula (403), and the conjugated siRNA sequence is , which corresponds to the sequence of complex 1 (i.e., L10-siC5a1M1SP) shown in Table 3. I did.

[0408] (Preparation Example 2) Preparation of Complexes 2 to 8 The sequences of the sense strand and the antisense strand are shown in Table 3, respectively. The sense and antisense strands corresponding to the siRNAs complexed in complex 4, complex 5, or complex 6 were The sequences shown in Table 3 were synthesized in the same manner as in Preparation Example 1, except that the sequences were the same as those in Preparation Example 1. The complexes 2 to 6 were synthesized and molecular weight detection was performed. Ta.

[0409] The sense and antisense strand sequences were respectively compounded with Complex 7 or Complex 8 shown in Table 3. The sense and antisense strands were synthesized to correspond to the siRNA that was synthesized. Except for the above, the same method as in Preparation Example 1 was used to synthesize the complexes 7 and 8 shown in Table 3, and the molecular weights were analyzed. However, the antisense strands of complexes 7 and 8 were the same as those of complex 1. Compared to the strand, it does not have a 5'-phosphate at the first nucleotide of the 5'-end. Therefore, in the process of preparing the antisense strand by the solid-phase phosphoramidite method, After binding the last nucleoside monomer of the antisense strand, the CPR-I monomer is bound. As a result, complexes 7 and 8 were obtained, respectively.

[0410] Table 3 shows the complex numbers and siRNA sequence compositions.

[0411] [Table 3]

[0412] (Preparation Example 3) Synthesis of siRNA sequence siRNA1 shown in Table 4a was synthesized in the same manner as in Preparation Example 1, except for the following two points. did. 1) For the sense strand, a general-purpose solid support (UnyLinker TM loaded NittoPhase(R) HL Solid Supports, Kinova The mixture was circulated using a starting material (Dietary Life Sciences). 2) For the antisense strand, the sequence of siRNA1 was complexed with complex 1. Compared with the antisense strand sequence of A, the first nucleotide at the 5'-end of siRNA1 There is no 5'-phosphate. Therefore, the antisense strand was prepared by the solid-phase phosphoramidite method. In the process of preparing the CP There is no need to bind the RI monomer. As a result, siRNA1 was obtained.

[0413] The sequences of the sense and antisense strands of the siRNA are shown in Table 4a. A2, the sense strand corresponding to siRNA3, siRNA4, siRNA5 or siRNA6 The siRNA was synthesized in the same manner as siRNA1, except that the siRNA was synthesized so that the sequence of the siRNA was the same as the antisense strand. siRNAs 2 to 6 were synthesized by the method described above. As a result, siRNAs 2 to 6 were obtained, respectively. .

[0414] Table 4a shows the siRNA numbers and siRNA sequence compositions.

[0415] [Table 4a]

[0416] (Preparation Example 4) Synthesis of Cy5-labeled siRNA complex and Cy5-labeled siRNA (4-1) Synthesis of Cy5-conjugate 1 and Cy5-conjugate 2 Cy5-conjugate 1 shown in Table 4b was synthesized by the same method as in Preparation Example 1, and molecular weight detection was performed. The sequences of the sense and antisense strands were as shown in Table 4b. The sense and antisense strands corresponding to the siRNA conjugated to Cy5-conjugate 1 are shown. The sequences were synthesized as follows: (1) siRNA conjugated to Cy5-conjugate 1 Since the Cy5 fluorescent group is covalently bonded to the 5' end of the sense strand of In the process of preparing the sense strand by the solid-phase phosphoramidite method described in step (1-2), After the last nucleoside monomer of the strand is attached, it is deprotected, coupled, capped, and The four reactions, namely, oxidation, and oxidation, were carried out to obtain the Cy5 phosphoramidite monomer (Shanghai Zhaowei Science and Technology Development Co., Ltd.). Co., Ltd., No. OP-057) must be attached to the 5' end of the sense strand. 2) The first nucleotide at the 5' end of the antisense strand of the siRNA conjugated to Cy5-conjugate 1 Since the nucleotide does not have a 5'-phosphate, the solid-phase phosphorylation described in step (1-3) of Preparation Example 1 was performed. In the process of preparing the antisense strand by the amidite method, the last nucleotide of the antisense strand is After coupling of the oside monomer, there is no need to couple the CPR-I monomer.

[0417] [ka] In the process of attaching the Cy5 phosphoramidite monomer to the 5' end of the sense strand, The conditions for the deprotection, coupling, capping, and oxidation reactions were as follows: 1) deprotection reaction time 2) Cy5 coupling reaction time was extended to 900 seconds. Except for this, the procedure was the same as in Preparation Example 1, step (1-2) synthesis of the sense strand.

[0418] Then, the sense strand was cleaved and deprotected under the following conditions. The combined nucleotide sequences were then soaked in AMA solution (40 wt% methylamine aqueous solution and 25 wt% amine aqueous solution). A 1:1 volumetric mixture of AMA solution and 0.5 ml / μm The mixture was incubated at 25°C for 2 hours in a water bath, and the remaining carrier was removed by filtration. The supernatant was then dried. The purification and desalting conditions for the sense strand were the same as those in Preparation Example 1, step (1-2) Synthesis of the sense strand. The purification and desalting conditions were the same as those of the previous study. Then, the mixture was lyophilized to obtain the sense strand of Cy5-conjugate 1. Ta.

[0419] As a result, Cy5-complex 1 was obtained, and the siRNA complex was A Cy5 fluorescent group was covalently attached to the 5' end of the fluorophore strand, and the Cy5-conjugate shown in Table 4b was used. It has sense and antisense strand sequences corresponding to those of target gene 1.

[0420] The sense and antisense strand sequences were conjugated to Cy5-conjugate 2, respectively, as shown in Table 4b. The sense and antisense strands were synthesized to correspond to the siRNA sequences. Cy5-conjugate 2 was prepared in the same manner as Cy5-conjugate 1, except for the following: This resulted in Cy5-conjugate 2.

[0421] (4-2) Synthesis of Cy5-siRNA1 and Cy5-siRNA2 General-purpose solid support (UnyLinker TM Loaded NittoPhase (registered trademark) HL Solid Supports, Kinovate Life Scie The preparation was carried out in the same manner as for Cy5-conjugate 1, except that Cy5-conjugate 1 (Nexes) was used as the starting material. Cy5-siRNA1 was prepared from the above and molecular weight detection was performed. Got A1.

[0422] The sense and antisense strand sequences correspond to Cy5-siRNA2 shown in Table 4b, respectively. Cy5-s was synthesized to have the corresponding sense and antisense sequences. Cy5-siRNA2 was prepared in the same manner as for iRNA1, and molecular weight detection was performed. This resulted in Cy5-siRNA2.

[0423] Table 4b shows Cy5-conjugate 1, Cy5-conjugate 2, Cy5-siRNA1 and Cy5- The siRNA2 numbers and siRNA sequences are indicated.

[0424] [Table 4b]

[0425] (Experimental Example 1) IgM inhibition of C5 mRNA in HepG2 cells by siRNA complex C 50 Measurement 10% fetal bovine serum (FBS, RMBIO Co., Ltd.) and 0.2% by volume of penicillin Treptomycin (Penicillin-Streptomycin, HyClone HepG2 cells (Nanjing Bao Biological Products) were cultured in H-DMEM complete medium (HyClone) containing (purchased from Eppendorf Technology Co., Ltd.) in a cell incubator containing 5% CO2 / 95% air at 37°C. The cells were then cultured in a broth.

[0426] 7 × 10 HepG2 cells 4 Cells / well were seeded into 24-well plates and incubated for 16 h. When the cell growth density reached 70-80%, remove all of the H-DMEM complete medium from the culture well. Remove the medium by suction and add 500 μl of Opti-MEM medium (GIBCO) to each well. The incubation was continued for 1.5 h.

[0427] DEPC-treated water was used to detect complex 1, complex 2, complex 3, complex 4, complex 5, and complex 6, complex 7 and complex 8, with concentrations of 20 μM, 5 μM, 1.25 μM, 0.313 μM M, 0.0781 μM, 0.0195 μM, and 0.0049 μM (as the amount of siRNA) A total of seven types of complex dilution standard solutions were prepared.

[0428] For each complex, solutions A1 to A7 were prepared, and each of the solutions A1 to A7 was prepared using the above seven types of and 50 μl of Opti-MEM medium in that order.

[0429] Solution B was prepared for each complex. Each solution B was prepared using Lipofectamine. e TM Contains 1 μl of 2000 and 50 μl of Opti-MEM medium.

[0430] For each complex, one B solution was mixed with one A1 to A7 solution in sequence, and the mixture was then The cells were then cultured at room temperature for 20 minutes to obtain transfection complexes X1 to X7. Two fusion complexes were prepared.

[0431] Mix one of the B solutions with 50 μl of Opti-MEM medium and incubate at room temperature for 20 min. Thus, transfection complex X8 was obtained. Four transfection complexes were prepared. did.

[0432] The above-mentioned cultured HepG2 cells were cultured in wells, and each complex was added at a volume of 100 μl / well. The corresponding transfection complexes X1 to X7 were added and mixed uniformly. The final concentrations (as siRNA) were 100 nM, 25 nM, 6.25 nM, and 1. The transfection concentrations were 56 nM, 0.391 nM, 0.098 nM, and 0.024 nM. The transfection mixture was obtained by mixing two transfection complexes X1 to X7 with the same concentration. The transfection mixture containing the complex was added to two different culture wells, and the test group The numbers ranged from 1 to 7.

[0433] In four separate culture wells, one transfection was performed at 100 μl / well. Complex X8 was added to each of the transfection mixtures to obtain a complex-free transfection mixture, and a control group was used. It was decided.

[0434] The transfection mixture containing the above complex and the transfection mixture without the complex were The mixture was incubated with the cells in the culture wells for 4 h, and then each well was diluted with 20% FBS-containing 1 ml of H-DMEM complete medium was added. The 24-well plate was incubated in 5% CO2 / 95% air. The cells were then placed in an air-containing cell incubator and cultured for 24 hours.

[0435] After that, RNAVzol (Vigorous Biotechnology (Beijing)) The total RNA was extracted using a 100% pure 100% ... Total RNA was extracted from the cells in each well.

[0436] For each well of cells, 1 μg of total RNA was used as a template for reverse transcription using the reverse transcription kit. Goldenstar TM RT6 cDNA Synthesis Kit (Beijing Ke The reagents were purchased from Yike New Biotechnology Co., Ltd. (No. TSK301M). Oldenstar TM Oligo (dT) 17 Select as primer and use kit According to the reverse transcription procedure in the manufacturer's manual, 20 μl of the reverse transcription reaction system was placed in each well. Reverse transcription was performed on total RNA of cells. The reverse transcription reaction system was set at 50°C. The reaction was incubated at 85°C for 50 min, then at 85°C for 5 min, and finally at 4°C for 30 s. After the reaction was completed, the To the transcription reaction system, 80 μl of DEPC-treated water was added to obtain a solution containing cDNA.

[0437] For each reverse transcription reaction system, 5 μl of the solution containing the above cDNA was used as a template for qPCR. NovoStart® SYBR qPCR SuperMix Plus The test kit was purchased from Kinki Protein Technology Co., Ltd. (No. E096-01B). A 20 μl qPCR reaction system was prepared using the reagent. Here, the target gene C5 and the endogenous reference The PCR primer sequences for amplifying the GAPDH gene are shown in Table 5. The final concentration was 0.25 μM. qPCR reactions were performed using ABI StepOnePlus The PCR product was placed in a Real-Time PCR device and amplified in a three-step manner. Pre-denaturation at 95°C for 10 min, then denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and 72°C for 10 min. The denaturation, annealing, and extension processes were repeated a total of 40 times, and the amplified DNA was The product W, which contains the target gene C5 and the endogenous reference gene GAPDH, was obtained. The composition W was incubated at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s in sequence, and the real-time The target gene C5 and the endogenous reference gene C5 were detected in product W by a fluorescent quantitative PCR system. A melting curve of the gene GAPDH was obtained, and the C5 of the target gene and the endogenous reference gene GAPDH were measured. t-values ​​were obtained.

[0438] [Table 5]

[0439] Using the comparative Ct (ΔΔCt) method, the expression level of the target gene C5 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 four culture wells in the control group. t (control group). Therefore, each culture well in each test group was ΔΔCt (test group), and each culture well in the control group had one ΔΔCt ( The control group corresponds to the control group.

[0440] The expression levels of C5 mRNA in the test group were normalized to those in the control group. The mRNA expression level was defined as 100%.

[0441] Relative expression level of C5 mRNA in the test group = 2^(-ΔΔCt(test group)) × 100% It is.

[0442] After transfection of different complexes (complexes 1–8), the expression of β-lactamase in HepG2 cells was According to the relative expression levels of C5 mRNA, the results were analyzed using Graphpad 5.0 software. Nonlinear regression analysis of log(inhibitor) vs. response e (three parameters) dose-effect curve fitting, Figure 1A Dose-effect curves of complexes 1 to 8 shown in Figures 1H to 1H were obtained. The logarithm of the final concentration (lgnM) is plotted on the abscissa and the relative expression level (%) of C5 mRNA is plotted on the ordinate. The coordinates are shown, and each black circle represents the C5 mRNA expression in two culture wells of the test group relative to the control group. The average relative expression level of A is shown.

[0443] The C of each complex was calculated from the following function corresponding to the fitted dose-effect curve: IC for 5 mRNA 50 The value was calculated.

[0444]

number

[0445] From the dose-effect curve and the corresponding function, the corresponding X when Y=50% is 50 value Determine the IC of each complex 50 Value=10^X 50 (nM) was calculated.

[0446] IC of each complex against C5 mRNA 50 Values ​​and R of fitted curves 2 value are summarized in Table 6.

[0447] [Table 6]

[0448] As can be seen from the results of Figures 1A to 1H and Table 6 above, the siRNPs provided by the present disclosure The A complex has high inhibitory activity in HepG2 liver cancer cells in vitro and IC 50 is 1.494 ~9.688nM.

[0449] (Experimental Example 2) Distribution of siRNA complexes in various organs of C57 mice Cy5-siRNA1, Cy5-complex1, Cy5-siRNA in 1x PBS solution 2 or Cy5-complex 2 in a solution at a concentration of 0.6 mg / ml (as siRNA). Dissolved.

[0450] Six to seven week-old C57BL / 6J female mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) Ten mice (simply referred to as C57 mice) were selected and randomly divided into groups of two. 1x PBS and the above-prepared Cy5-siRNA1, Cy5-complex 1, and Cy5- siRNA2 or Cy5-complex 2 solution was injected subcutaneously, and all animals were dosed according to body weight. The dose was calculated and the administration volume was 5 ml / kg body weight, and the amount of siRNA was The corresponding dose was 3 mg / kg body weight.

[0451] After 2, 6, 24, and 48 hours from administration, the mice in each group were sequentially examined using small animal biooptical imaging. The system was placed in an IVIS Lumina Series III and was then fitted with isoflurane. The mouse was anesthetized using gas, and the anesthetized mouse was placed in a belly-up position for small animal biooptical imaging. The cells were placed in a gauging system and subjected to live imaging to dynamically detect Cy5 fluorescent signals and The distribution of the y5-labeled siRNA or Cy5-labeled complex in living animals was tracked. Only mice administered the sensitizing complex showed a significantly enhanced fluorescent signal in the liver region, and Mice administered y5-labeled siRNA or 1×PBS did not show any fluorescent signal in the liver region. No number was given.

[0452] After 48 h, all mice were killed and five tissues from each mouse, namely the heart, lungs, liver, spleen, and kidneys, were extracted. The organs were then extracted and fluorescent images were taken with the IVIS Lumina Series III. One mouse was selected from each group, and the above five organs were arranged vertically in order. × PBS, Cy5-siRNA1, or Cy5-complex 1 administered mice were identified. The results are shown in Fig. 2A. The organs of mice administered 5-complex 2 were imaged in the same field of view, and the results are shown in Figure 2B. .

[0453] As can be seen from Figures 2A and 2B, only Cy5-conjugate 1 and Cy5-conjugate 2 were detected in the liver. Cy5-conjugate 1 and Cy5-conjugate 2 accumulated in small amounts in the kidney. The complex of the present disclosure effectively and specifically delivers siRNA to the liver, whereas it does not accumulate in other organs. Furthermore, Figure 1G (complex 7) showed high in vitro inhibitory activity. ) and Fig. 1F (which showed that complex 6 had high in vitro inhibitory activity) As a result, the complex provided by the present disclosure specifically suppresses the expression of target genes in the liver. He suggested that it was possible.

[0454] Although several embodiments of the present disclosure have been described in detail above, the present disclosure The present disclosure is not limited to the specific details, but is within the scope of the technical idea of ​​the present disclosure. A number of simple modifications can be made to the present disclosure, all of which are within the scope of the present disclosure. Belongs to the range.

[0455] In addition, it should be noted that each of the specific techniques described in the above embodiments Technical features may be combined in any suitable manner, provided there is no contradiction, and To avoid unnecessary duplication, this disclosure will not separately describe the various possible combination methods.

[0456] In addition, various different embodiments of the present disclosure may be combined in any combination, and the spirit of the present disclosure will be apparent to those skilled in the art. Unless otherwise stated, such combinations should also be considered as disclosed in this disclosure. be.< / sirna>

Claims

1. An siRNA conjugate having a structure as shown in formula (308): 【Chemistry 1】 During the ceremony, n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4. m1, m2, or m3 is independently an integer selected from 2 to 10; R １ ０ , R １１ , R １２ , R １３ , R １４ Or R １５ are each independently H; or , C １ -C １０ Alkyl group, C １ -C １０ Halogenated alkyl group and C １ -C １０ Arco oxy groups; R ３ is a group having the structure shown in formula A59, 【Chemistry 2】 In the formula, E １ is OH, SH or BH ２ and Nu is an siRNA, the siRNA comprises a sense strand and an antisense strand, Each nucleotide of the iRNA is independently a modified or unmodified nucleotide, The sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially inverted. The nucleotide sequence I and the nucleotide sequence II form a double-stranded region complementary to each other. It is one selected from the sequences shown in i) to vi) 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'-CUUCAUUCAUACAGACAAZ １ -3' (SEQ ID NO:1), 5'-Z ２ UUGUCUGUAUGAAUGAAG-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 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'-CUACAGUUUAGAAGAUUUZ ５ -3' (SEQ ID NO:61), 5'-Z ６ AAACUUCUAAACUGUAG-3' (SEQ ID NO:62) However, Z ５ is A and Z ６ is U, 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'-GGAAGGUUACCGAGCAAUZ ９ -3' (SEQ ID NO: 121), 5'-Z １０ AUUGCUCGGUAACCUUCC-3 '(SEQ ID NO: 122) However, Z ９ is A and Z １０ is U, 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'-AGAACAGACAGCAGAAUUZ １３ -3' (SEQ ID NO: 181), 5'-Z １４ AAUUCUGCUGUCUGUUCU-3 '(SEQ ID NO: 182) However, Z １３ is A and Z １４ is U, 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'-CCAAGAAGAACGCUGCAAZ １７ -3' (SEQ ID NO: 241), 5'-Z １８ UUGCAGCGUUCUUCUUGG-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'-CCAGUAAGCAAGCCAGAAZ ２１ -3' (SEQ ID NO: 301), 5'-Z ２２ UUCUGGCUUGCUUACUGG-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 R ２ is a straight chain alkylene group of 1 to 20 carbon atoms in length, where one or more 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 is 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, has multiple carbon atoms, C(O), NH, O, S, CH=N, S(O) ２ , C ２ -C １０ a Alkenylene group, C ２ -C １０ Alkynylene group, C ６ -C １０ Arylene group, C ３ -C １８ Heterocyclylene group and C ５ -C １０ Heteroarylene groups, is optionally substituted with multiple groups; L １ is C １ -C １０ Alkyl group, C ６ -C １０ Aryl groups, C ５ -C １０ Heteroaryl group, C １ -C １０ Halogenated alkyl group, -OC １ -C １０ 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 Alkylphenyl group, -C １ -C １０ Alkyl-SH, -SC １ -C １０ Al halide alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ -C １０ Alkyl-NH ２ , -N(C １ -C １０ Alkyl group) (C １ -C １０ alkyl group), -NH(C １ -C １０ alkyl group), -N(C １ -C １０ Alkyl group) (C １ -C １０ alkylphenyl group), -NH(C １ -C １０ alkylphenyl group), cyano group, nitro group, -CO ２ H, -C ( O) O (C １ -C １０ alkyl group), -CON(C １ -C １０ Alkyl group) (C １ -C １ ０ 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 a 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 Al alkylphenyl group, -C(O)C １ -C １０ Haloalkyl groups, —OC(O)C １ -C １０ a 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 a 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) and optionally having one or more substituents selected from the group consisting of: 【change】 represents the site at which a group is covalently attached, M １ represents the targeting group, siRNA conjugate.

2. Each L １ independently selected from the group consisting of (A1) to (A26) groups and any bond combinations thereof Selected, 【Chemistry 3】 wherein each j1 is independently an integer from 1 to 20, and each j2 is independently an integer from 1 to 20. the law of nature, Each R' is independently C １ -C １０ is an alkyl group, Each Ra is a group represented by formula (A27) to (A45) or any combination of bonds thereof. is selected from the group consisting of 【Chemistry 4】 Each Rb is independently C １ -C １０ is an alkyl group, Or, L １ is selected from A1, A4, A5, A6, A8, A10, A11, and A13 and a combination of one or more bonds, Or, L １ At least two selected from A1, A4, A8, A10 and A11 It is a combination of bonds, Or, L １ is a combination of at least two bonds selected from A1, A8, and A10; the law of nature, Or, L １ is 3 to 25 atoms in length, Or, L １ is 4 to 15 atoms in length, 【change】 The siRNA conjugate of claim 1 , wherein represents the site to which the group is covalently attached.

3. 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 is C １ -C ５ is an alkyl group, Alternatively, j1 is an integer from 3 to 5, j2 is an integer from 3 to 5, and R' is a methyl group. Ra is one of a group represented by formula (A27) or a group represented by formula (A28). A28), where Rb is a methyl group, an ethyl group, an isopropyl group, or a butyl group. The siRNA conjugate of claim 2 , which is one of the groups.

4. 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 1 to 3.

5. Each m1, m2 or m3 is independently an integer from 2 to 5, and / or m1=m The siRNA complex of any one of claims 1 to 4, wherein m2=m3.

6. Each of said targeting groups independently binds to an asialoglycoprotein receptor on the surface of mammalian hepatocytes. is a ligand having affinity for Alternatively, each said targeting group is independently an asialoglycoprotein or a sugar; Alternatively, each of the targeting groups is selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose D-galactose, L-galactose, α-D-mannofuranose, β-D-mannose Furanose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose Ranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranos α-D-fructofuranose, α-D-fructopyranose, α-D-galactose Pyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactopyranose Citrobranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine Galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N- n-Butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, Oxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formaldehyde Mido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamine No-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucose Leucopyranose, methyl 2,3,4-tris-O-acetyl-1-thio-6-O-trityl 4-Thio-β-D-galactopyranose, ethyl 3,4 ,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptide Topiranoside, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D- One independently selected from 4-thioribose, L-ribose, and L-4-thioribose can be, Alternatively, at least one or each of the targeting groups is galactose or N-acetylgalactose. The siRNA conjugate of any one of claims 1 to 5, wherein the siRNA conjugate is tosamine.

7. R １０ , R １１ , R １２ , R １３ , R １４ Or R １５ are each independently H, a phenyl group or an ethyl group, Or, R １０ , R １１ , R １２ , R １３ , R １４ Or R １５ are both H. The siRNA complex according to any one of claims 1 to 6.

8. R ２ The nitrogen-containing backbone is bonded to the N atom and R ３ is bonded to a P atom in Both parts are included, Or, R ２ the site bonded to N on the nitrogen-containing backbone forms an amide bond with N; The R ３ The site bonded to the P atom in Or, R ２ is selected from the groups represented by formula (B5), (B6), (B5') or (B6'). The siRNA conjugate according to any one of claims 1 to 7, wherein

9. Equations (403), (404), (405), (406), (407), (408), (4 ０９）、（４１０）、（４１１）、（４１２）、（４１３）、（４１４）、（４１５）、 (416), (417), (418), (419), (420), (421) or (42 2) The siRNA complex according to any one of claims 1 to 8, having the structure shown in

10. The P atom in formula A59 is attached to the end of the sense 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. Refers to cleotide, Alternatively, the P atom in formula A59 is a cyclic alkyl group of the sense or antisense strand of the siRNA. or the P atom in formula A59 is bonded to the 3' end of the sense strand of the siRNA. is combined with Alternatively, the P atom in formula A59 is linked to the siRNA via a phosphodiester bond. Any one of claims 1 to 9, which is bound to the 2', 3' or 5' position of a nucleotide in Item 1. The siRNA complex according to item 1.

11. i) a nucleotide sequence between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1; and / or the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 have no more than one octide difference. or ii) a nucleotide sequence between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61; and / or the nucleotide sequence II and SEQ ID NO:62 have no more than one nucleotide difference. or iii) between said 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, or 、 iv) between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 and / or said nucleotide sequence II and SEQ ID NO:1 have no more than one nucleotide difference. 82 has no more than one nucleotide difference between it and the nucleotide sequence shown in v) a nucleotide sequence between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:241; and / or the nucleotide sequence II and SEQ ID NO:24 have no more than one nucleotide difference. 2 has no more than one nucleotide difference between the nucleotide sequence shown in vi) between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301; and / or said nucleotide sequence II and SEQ ID NO:3 have no more than one nucleotide difference.

1. There is no more than one nucleotide difference between the nucleotide sequence shown in The siRNA complex described in

12. i) a nucleic acid sequence between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2; The difference between the leotide and Z ４ Including the difference at the position of Z ４ is selected from A, C, or G; or 、 ii) between said 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 A, C, or G; or Yes, iii) the sequence of nucleotide sequence II with the nucleotide sequence shown in SEQ ID NO: 122 The nucleotide difference between Z １２ Including the difference at the position of Z １２ Select from A, C or G Or, iv) between said 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 A, C, or G Or, v) between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 The nucleotide difference is Z ２０ Including the difference at the position of Z ２０ is selected from A, C, or G , or vi) between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 302 The nucleotide difference between ２４ Including the difference at the position of Z ２４ is selected from A, C, or G The siRNA complex of claim 1 or 11.

13. Z ３ Z ４ or Z ７ Z ８ and complementary nucleotide or Z １１ Z １２ or Z １５ Z １６ or Z １９ Z ２０ with the complementary nucleotide Yes, or Z ２３ Z ２４ The si of claim 12 is a nucleotide complementary to RNA complexes.

14. The nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially Reverse complementary or completely reverse complementary, and essentially reverse complementary means that the two nucleotide sequences are The term "substantially reverse complementary" refers to a sequence having three or fewer base mismatches between the two sequences. This refers to the presence of one or less base mismatches between the two nucleotide sequences. Reverse complementary means that there is no mismatch between the two nucleotide sequences.

4. The siRNA complex according to any one of claims 3.

15. 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 less base mismatches between nucleotide sequences. Complementary means that there is no mismatch between the two nucleotide sequences.

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

16. i) 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, The nucleotide sequences III and IV are each one nucleotide in length, The base of III is U, or the nucleotide sequences III and IV are both 2 nucleotides, from the 5' end to the 3' end, The base sequence is CU, or the nucleotide sequences III and IV are both 3 nucleotides in length. nucleotide having the nucleotide sequence of nucleotide III from the 5' end to the 3' end. The sequence is UCU, or the nucleotide sequences III and IV are each 4 nucleotides in length. nucleotide sequence III from the 5' end to the 3' end is UUCU, or ii) said nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:63; The nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:64, Nucleotide sequences III and IV are each one nucleotide in length, The base of nucleotide sequence III is A, or the nucleotide sequences III and IV are different in length. Each of the sequences is 2 nucleotides. From the 5' end to the 3' end, the sequence is The base sequence of I is UA, or the nucleotide sequences III and IV are both From the 5' end to the 3' end, the nucleotide sequence III is The base sequence is AUA, or the nucleotide sequences III and IV are both 4 nucleotides, from the 5' end to the 3' end, The base sequence is CAUA, or iii) said 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; The nucleotide sequences III and IV are each one nucleotide in length. The base of nucleotide sequence III is G, or the nucleotide sequences III and IV are The length of each is 2 nucleotides, and the nucleotide sequence from the 5' end to the 3' end is The base sequence of sequence III is AG, or the nucleotide sequences III and IV have a length Each of them is a trinucleotide, and from the 5' end to the 3' end, it is nucleotide sequence I The base sequence of II is CAG, or the nucleotide sequences III and IV are different in length. Each of the sequences is 4 nucleotides, and from the 5' end to the 3' end, the sequence is The base sequence of I is CCAG, or iv) said nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 183; wherein the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 184; The nucleotide sequences III and IV are each one nucleotide in length. the base of nucleotide sequence III is G; or the nucleotide sequences III and IV have a length Each of these is two nucleotides, and from the 5' end to the 3' end, the nucleotide sequence The base sequence of III is GG, or the nucleotide sequences III and IV are different in length. Each of the sequences is 3 nucleotides, and from the 5' end to the 3' end, the sequence is The base sequence of I is AGG, or the nucleotide sequences III and IV are of equal length. Each of these is a 4-nucleotide sequence, and from the 5' to the 3' end, the sequence is is CAGG, or v) said nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 243; The nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 244, Nucleotide sequences III and IV are each one nucleotide in length, the base of nucleotide sequence III is G; or the nucleotide sequences III and IV have a length Each of them is two nucleotides, and from the 5' end to the 3' end, it is nucleotide sequence I The base sequence of II is GG, or the nucleotide sequences III and IV are of equal length. Each of these is a trinucleotide and, from the 5' end to the 3' end, has the nucleotide sequence III or the nucleotide sequence III and IV are both From the 5' end to the 3' end, the nucleotide sequence III is The base sequence is CAGG, or vi) said nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303; wherein the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 304; The nucleotide sequences III and IV are each one nucleotide in length. the base of nucleotide sequence III is A; or the nucleotide sequences III and IV have a length Each of these is two nucleotides, and from the 5' end to the 3' end, the nucleotide sequence The base sequence of III is UA, or the nucleotide sequences III and IV are different in length. Each of the sequences is 3 nucleotides, and from the 5' end to the 3' end, the sequence is The base sequence of I is UUA, or the nucleotide sequences III and IV are the same in length. Each of these is a 4-nucleotide sequence, and from the 5' to the 3' end, the sequence is The siRNA complex of claim 15, wherein the base sequence is GUUA.

17. 17. The method of claim 16, wherein the nucleotide sequences III and IV are completely reverse complementary. siRNA complex.

18. 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. or said nucleotide sequence V is 2 in length. Alternatively, the nucleotide sequence V is a sequence of two consecutive thymine deoxyribonuclease (TDM) residues. a uracil ribonucleotide or two consecutive uracil ribonucleotides, or The nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. The siRNA complex according to any one of claims 1 to 17.

19. The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:5, The antisense strand comprises the nucleotide sequence shown in SEQ ID NO:6, or The sense strand of A comprises the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand is , comprising the nucleotide sequence shown in SEQ ID NO:8, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

65. and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:66; or The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:67, the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:68, 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 shown in SEQ ID NO:

126. or the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

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

128. Contains the array of Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

185. wherein the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 186; or wherein the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 187; the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 188; 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. or the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

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

248. Contains the array of 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. or the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

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

308. The siRNA conjugate of any one of claims 1 to 18, comprising the sequence:

20. The siRNA is siC5a1, siC5a2, siC5b1, siC5b2, si C5c1, siC5c2, siC5d1, siC5d2, siC5e1, siC5e2, 19. The nucleotide sequence of siC5f1 or siC5f2 according to claims 11 to 19.

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

21. 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 conjugate of any one of claims 1 to 20, which is an ester group.

22. Each nucleotide in the sense strand and the antisense strand is independently fluoro-modified. nucleotide or a non-fluoro modified nucleotide, Alternatively, the fluoro-modified nucleotide is a nucleotide sequence of nucleotide sequence I I, from the 5' end to the 3' end, at least the 1st base of said nucleotide sequence I The 7th, 8th and 9th nucleotides are fluoro-modified nucleotides, and the 5' to 3' nucleotides are At least the 2nd, 6th, 14th and 16th nucleotides of said nucleotide sequence II are tide is a fluoro-modified nucleotide, Alternatively, 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 of sequence I are fluoro-modified nucleotides. and the remaining nucleotides in the sense strand are non-fluoro-modified nucleotides. and in the antisense strand, the nucleotide sequence is Nucleotides at positions 2, 6, 14, and 16 or 2, 6, 8, 9, 14, and 16 of nucleotide sequence II and the nucleotides at the remaining positions in the antisense strand are fluoro-modified nucleotides. The siRNA conjugate of claim 21 , wherein the nucleotide is a non-fluoro-modified nucleotide.

23. 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. This is one of the Alternatively, the hydroxy group at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorine group. The nucleotide may be a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2' -amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxy nucleotides nucleotide analogs are selected from isonucleotides, LNAs, one selected from ENA, cET, UNA and GNA; Alternatively, each non-fluoro-modified nucleotide is a methoxy-modified nucleotide. The methoxy-modified nucleotide is a nucleotide in which the 2'-hydroxy group of the ribose group is replaced with methoxy.

23. The siRNA conjugate of claim 21 or 22, wherein the nucleotide is a substituted nucleotide.

24. 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. It is 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 complex according to any one of claims 21 to 23,

25. The siRNA is siC5a1-M1, siC5a2-M1, siC5a1-M2, siC5a2-M2, siC5a1-M3, siC5a2-M3, siC5b1-M1, siC5b2-M1, siC5b1-M2, siC5b2-M2, siC5b1-M3, siC5b2-M3, siC5c1-M1, siC5c2-M1, siC5c1-M2, siC5c2-M2, siC5c1-M3, siC5c2-M3, siC5d1-M1, siC5d2-M1, siC5d1-M2, siC5d2-M2, siC5d1-M3, siC5d2-M3, siC5e1-M1, siC5e2-M1, siC5e1-M2, siC5e2-M2, siC5e1-M3, siC5e2-M3, siC5f1-M1, siC5f2-M1, siC5f1-M2, siC5f2-M2, siC5f1-M3 or is any one of siC5f2-M3 according to any one of claims 1 to 24. iRNA complexes.

26. The phosphate ester group having the modifying group is a phosphate diester in the phosphate ester group. a thiophosphate group in which at least one oxygen atom of the bond is replaced with a sulfur atom; Alternatively, the phosphate ester group having the modifying group is a thiol having a structure represented by formula (1): The siRNA conjugate of claim 21 , which is a phosphate group. 【Chemistry 5】

27. 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 s according to claim 26 is bonded to at least one selected from the group consisting of iRNA complexes.

28. The siRNA is siC5a1-M1S, siC5a2-M1S, siC5a1-M 2S, siC5a2-M2S, siC5a1-M3S, siC5a2-M3S, siC5 b1-M1S, siC5b2-M1S, siC5b1-M2S, siC5b2-M2S, siC5b1-M3S, siC5b2-M3S, siC5c1-M1S, siC5c2- M1S, siC5c1-M2S, siC5c2-M2S, siC5c1-M3S, siC 5c2-M3S, siC5d1-M1S, siC5d2-M1S, siC5d1-M2S , siC5d2-M2S, siC5d1-M3S, siC5d2-M3S, siC5e1 -M1S, siC5e2-M1S, siC5e1-M2S, siC5e2-M2S, si C5e1-M3S, siC5e2-M3S, siC5f1-M1S, siC5f2-M1 S, siC5f1-M2S, siC5f2-M2S, siC5f1-M3S or siC5 The siRNA according to any one of claims 1 to 27, which is any one of f2-M3S. Complex.

29. The 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide. or 5'-phosphate analog modified nucleotides, Alternatively, the 5'-phosphate nucleotide is a nucleotide having a structure shown in formula (2): The 5'-phosphate analog modified nucleotide is represented by any one of formulas (3) to (6). nucleotides selected from those having the structure shown in any one of 【Chemistry 6】 In the formula, R is selected from H, OH, a methoxy group, or fluorine, and Base represents a base. siR according to any one of claims 1 to 28, wherein the siR is selected from the group consisting of A, U, C, G and T. NA complex.

30. The siRNA is siC5a1-M1P1, siC5a1-M2P1, siC5a1 -M3P1, siC5a2-M1P1, siC5a2-M2P1, siC5a2-M3P 1, siC5a1-M1SP1, siC5a1-M2SP1, siC5a1-M3SP1 , siC5a2-M1SP1, siC5a2-M2SP1, siC5a2-M3SP1, siC5b1-M1P1, siC5b1-M2P1, siC5b1-M3P1, siC5 b2-M1P1, siC5b2-M2P1, siC5b2-M3P1, siC5b1-M 1SP1, siC5b1-M2SP1, siC5b1-M3SP1, siC5b2-M1 SP1, siC5b2-M2SP1, siC5b2-M3SP1, siC5c1-M1P 1, siC5c1-M2P1, siC5c1-M3P1, siC5c2-M1P1, si C5c2-M2P1, siC5c2-M3P1, siC5c1-M1SP1, siC5c 1-M2SP1, siC5c1-M3SP1, siC5c2-M1SP1, siC5c2 -M2SP1, siC5c2-M3SP1, siC5d1-M1P1, siC5d1-M 2P1, siC5d1-M3P1, siC5d2-M1P1, siC5d2-M2P1, siC5d2-M3P1, siC5d1-M1SP1, siC5d1-M2SP1, si C5d1-M3SP1, siC5d2-M1SP1, siC5d2-M2SP1, siC 5d2-M3SP1, siC5e1-M1P1, siC5e1-M2P1, siC5e1 -M3P1, siC5e2-M1P1, siC5e2-M2P1, siC5e2-M3P 1, siC5e1-M1SP1, siC5e1-M2SP1, siC5e1-M3SP1 , siC5e2-M1SP1, siC5e2-M2SP1, siC5e2-M3SP1, siC5f1-M1P1, siC5f1-M2P1, siC5f1-M3P1, siC5 f2-M1P1, siC5f2-M2P1, siC5f2-M3P1, siC5f1-M 1SP1, siC5f1-M2SP1, siC5f1-M3SP1, siC5f2-M1 SP1, siC5f2-M2SP1, or siC5f2-M3SP1. The siRNA complex according to any one of claims 1 to 29.

31. An siRNA comprising a sense strand and an antisense strand, Each nucleotide in the strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide. nucleotides, the sense strand comprising nucleotide sequence I, and the antisense strand comprising The nucleotide sequence I and the nucleotide sequence II are , forming a double-stranded region in at least a portion in a reverse complementary manner, and the fluoro-modified nucleotide is Located in nucleotide sequence I and nucleotide sequence II, from the 5' end to the 3' end In the sense strand, the nucleotides at positions 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 The nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluoro modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non- is a fluoro-modified nucleotide, 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 that are the same length and have no more than three nucleotide differences, In nucleotide sequence I, position Z １ Nucleotide Z corresponding to ３ The nucleoside is In sequence II, position Z ２ Nucleotide Z corresponding to ４ and Z ４ The above A the first nucleotide at the 5' end of the antisense strand, or 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. In said nucleotide sequence I, position Z ５ Nucleotide Z corresponding to ７ The above-mentioned In nucleotide sequence II, position Z ６ Nucleotide Z corresponding to ８ and Z ８ teeth, the first nucleotide at the 5' end of the antisense strand, or 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 in said nucleotide sequence I, position Z ９ Nucleotide Z corresponding to １１ Includes In said nucleotide sequence II, position Z １０ Nucleotide Z corresponding to １２ Includes: Said Z １２ is the first nucleotide at the 5' end of the antisense strand, or 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. and in said nucleotide sequence I, position Z １３ Nucleotide Z corresponding to １５ Includes In said nucleotide sequence II, position Z １４ Nucleotide Z corresponding to １６ Includes: Said Z １６ is the first nucleotide at the 5' end of the antisense strand, or 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. and in said nucleotide sequence I, position Z １７ Nucleotide Z corresponding to １９ Includes: In said nucleotide sequence II, position Z １８ Nucleotide Z corresponding to ２０ Contains, before Diary Z ２０ is the first nucleotide at the 5' end of the antisense strand, or 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. and in said nucleotide sequence I, position Z ２１ Nucleotide Z corresponding to ２３ Includes In said nucleotide sequence II, position Z ２２ Nucleotide Z corresponding to ２４ Includes: Said Z ２４ is the first nucleotide at the 5' end of the antisense strand, A.

32. 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 of claim 31 , which is one of the above.

33. 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 , cET, UNA, and GNA. 。

34. 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 31 to 33, which refers to a nucleotide.

35. i) a nucleotide sequence between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1; and / or the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2 have no more than one octide difference. or ii) a nucleotide sequence between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61; and / or the nucleotide sequence II and SEQ ID NO:62 have no more than one nucleotide difference. or iii) between said 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, or 、 iv) between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 181 and / or said nucleotide sequence II and SEQ ID NO:1 have no more than one nucleotide difference. 82 has no more than one nucleotide difference between it and the nucleotide sequence shown in v) a nucleotide sequence between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:241; and / or the nucleotide sequence II and SEQ ID NO:24 have no more than one nucleotide difference. 2 has no more than one nucleotide difference between the nucleotide sequence shown in vi) between said nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 301; and / or said nucleotide sequence II and SEQ ID NO:3 have no more than one nucleotide difference.

3. The nucleotide sequence of claim 2, which has no more than one nucleotide difference between the nucleotide sequence shown in 35. The siRNA according to any one of claims 1 to 34.

36. i) a nucleic acid sequence between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2; The difference between the leotide and Z ４ Including the difference at the position of Z ４ is selected from A, C, or G; or 、 ii) between said 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 A, C, or G; or Yes, iii) the sequence of nucleotide sequence II with the nucleotide sequence shown in SEQ ID NO: 122 The nucleotide difference between Z １２ Including the difference at the position of Z １２ Select from A, C or G Or, iv) between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 The nucleotide difference between １６ Including the difference at the position of Z １６ is selected from A, C, or G Or, v) between said nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 The nucleotide difference is Z ２０ Including the difference at the position of Z ２０ is selected from A, C, or G , or vi) between said 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 is selected from A, C, or G The siRNA according to any one of claims 31 to 35.

37. Z ３ But, Z ４ or Z ７ But, Z ８ and complementary nuclease or Z １１ But, Z １２ or Z １ ５ But, Z １６ or Z １９ But, Z ２０ and complementary Nuk or Z ２３ But, Z ２４ and is a nucleotide complementary to The siRNA described in

38. The nucleotide sequence I and the nucleotide sequence II are essentially reverse complementary, substantially Reverse complementary or completely reverse complementary, and essentially reverse complementary means that the two nucleotide sequences are The term "substantially reverse complementary" refers to a sequence having three or fewer base mismatches between the two sequences. This refers to the presence of one or less base mismatches between the two nucleotide sequences. Reverse complementary means that there is no mismatch between the two nucleotide sequences.

37. The siRNA according to any one of claims 37.

39. 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 less base mismatches between nucleotide sequences. Complementary means that there is no mismatch between the two nucleotide sequences. The siRNA according to any one of claims 1 to 5.

40. i) 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, The nucleotide sequences III and IV are each one nucleotide in length, The base of III is U, or the nucleotide sequences III and IV are both 2 nucleotides, from the 5' end to the 3' end, The base sequence is CU, or the nucleotide sequences III and IV are both 3 nucleotides in length. nucleotide having the nucleotide sequence of nucleotide III from the 5' end to the 3' end. The sequence is UCU, or the nucleotide sequences III and IV are each 4 nucleotides in length. nucleotide sequence III from the 5' end to the 3' end is UUCU, or ii) said nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:63; The nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:64, Nucleotide sequences III and IV are each one nucleotide in length, The base of nucleotide sequence III is A, or the nucleotide sequences III and IV are different in length. Each of the sequences is 2 nucleotides. From the 5' end to the 3' end, the sequence is The base sequence of I is UA, or the nucleotide sequences III and IV are both From the 5' end to the 3' end, the nucleotide sequence III is The base sequence is AUA, or the nucleotide sequences III and IV are both 4 nucleotides, from the 5' end to the 3' end, The base sequence is CAUA, or iii) said 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; The nucleotide sequences III and IV are each one nucleotide in length. The base of nucleotide sequence III is G, or the nucleotide sequences III and IV are The length of each is 2 nucleotides, and the nucleotide sequence from the 5' end to the 3' end is The base sequence of sequence III is AG, or the nucleotide sequences III and IV have a length Each of them is a trinucleotide, and from the 5' end to the 3' end, it is nucleotide sequence I The base sequence of II is CAG, or the nucleotide sequences III and IV are different in length. Each of the sequences is 4 nucleotides, and from the 5' end to the 3' end, the sequence is The base sequence of I is CCAG, or iv) said nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 183; wherein the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 184; The nucleotide sequences III and IV are each one nucleotide in length. the base of nucleotide sequence III is G; or the nucleotide sequences III and IV have a length Each of these is two nucleotides, and from the 5' end to the 3' end, the nucleotide sequence The base sequence of III is GG, or the nucleotide sequences III and IV are different in length. Each of the sequences is 3 nucleotides, and from the 5' end to the 3' end, the sequence is The base sequence of I is AGG, or the nucleotide sequences III and IV are of equal length. Each of these is a 4-nucleotide sequence, and from the 5' to the 3' end, the sequence is is CAGG, or v) said nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 243; The nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 244, Nucleotide sequences III and IV are each one nucleotide in length, the base of nucleotide sequence III is G; or the nucleotide sequences III and IV have a length Each of them is two nucleotides, and from the 5' end to the 3' end, it is nucleotide sequence I The base sequence of II is GG, or the nucleotide sequences III and IV are of equal length. Each of these is a trinucleotide and, from the 5' end to the 3' end, has the nucleotide sequence III or the nucleotide sequence III and IV are both From the 5' end to the 3' end, the nucleotide sequence III is The base sequence is CAGG, or vi) said nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303; wherein the nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 304; The nucleotide sequences III and IV are each one nucleotide in length. the base of nucleotide sequence III is A; or the nucleotide sequences III and IV have a length Each of these is two nucleotides, and from the 5' end to the 3' end, the nucleotide sequence The base sequence of III is UA, or the nucleotide sequences III and IV are different in length. Each of the sequences is 3 nucleotides, and from the 5' end to the 3' end, the sequence is The base sequence of I is UUA, or the nucleotide sequences III and IV are the same in length. Each of these is a 4-nucleotide sequence, and from the 5' to the 3' end, the sequence is The siRNA according to any one of claims 31 to 39, wherein the base sequence is GUUA.

41. The nucleotide sequences III and IV are completely reverse complementary to each other. RNA.

42. 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. or said nucleotide sequence V is 2 in length. Alternatively, the nucleotide sequence V is a sequence of two consecutive thymine deoxyribonuclease (TDM) residues. a uracil ribonucleotide or two consecutive uracil ribonucleotides, or The nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.

42. The siRNA according to any one of claims 31 to 41.

43. The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:5, The antisense strand comprises the nucleotide sequence shown in SEQ ID NO:6, or The sense strand of A comprises the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand is , comprising the nucleotide sequence shown in SEQ ID NO:8, Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

65. and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:66; or The sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:67, the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:68, 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 shown in SEQ ID NO:

126. or the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

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

128. Contains the array of Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:

185. wherein the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 186; or wherein the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 187; the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 188; 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. or the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

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

248. Contains the array of 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. or the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:

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

308. The siRNA of any one of claims 31 to 42, comprising the sequence:

44. siC5a1, siC5a2, siC5b1, siC5b2, siC5c1, siC5 c2, siC5d1, siC5d2, siC5e1, siC5e2, siC5f1 or s 44. The method according to any one of claims 31 to 43, having the nucleotide sequence shown in iC5f2. The siRNA described above.

45. siC5a1-M1, siC5a2-M1, siC5b1-M1, siC5b2-M1 , siC5c1-M1, siC5c2-M1, siC5d1-M1, siC5d2-M1 , siC5e1-M1, siC5e2-M1, siC5f1-M1 or siC5f2-M The siRNA according to any one of claims 31 to 44, which is any one of the above.

46. At least one phosphate group in the sense strand or the antisense strand is modified The siRNP of any one of claims 31 to 45, which is a phosphate ester group having a group. A.

47. The phosphate ester group having the modifying group is a phosphate diester in the phosphate ester group. a thiophosphate group in which at least one oxygen atom of the bond is replaced with a sulfur atom; Alternatively, the phosphate group having the modifying group may be a thiol having the structure shown in formula (1): The siRNA of claim 46, which is a phosphate group. 【Chemistry 7】

48. 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 s of claim 47 is present bound to at least one selected from the group consisting of iRNA.

49. The siRNA is siC5a1-M1S, siC5a2-M1S, siC5b1-M 1S, siC5b2-M1S, siC5c1-M1S, siC5c2-M1S, siC5 d1-M1S, siC5d2-M1S, siC5e1-M1S, siC5e2-M1S, siC5f1-M1S or siC5f2-M1S, 9. An siRNA according to any one of claims 8 to 8.

50. The 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide. or 5'-phosphate analog modified nucleotides, Alternatively, the 5'-phosphate nucleotide is a nucleotide having a structure shown in formula (2): The 5'-phosphate analog modified nucleotide is represented by any one of formulas (3) to (6). nucleotides selected from those having the structure shown in any one of 【Chemistry 8】 In the formula, R is selected from H, OH, a methoxy group, or fluorine, and Base represents a base.

50. The si according to any one of claims 31 to 49, wherein the si is selected from the group consisting of A, U, C, G and T. RNA.

51. siC5a1-M1P1, siC5a2-M1P1, siC5a1-M1SP1, si C5a2-M1SP1, siC5b1-M1P1, siC5b2-M1P1, siC5b 1-M1SP1, siC5b2-M1SP1, siC5c1-M1P1, siC5c2- M1P1, siC5c1-M1SP1, siC5c2-M1SP1, siC5d1-M1 P1, siC5d2-M1P1, siC5d1-M1SP1, siC5d2-M1SP1 , siC5e1-M1P1, siC5e2-M1P1, siC5e1-M1SP1, si C5e2-M1SP1, siC5f1-M1P1, siC5f2-M1P1, siC5f siC5f2-M1SP1 or siC5f2-M1SP1. The siRNA according to any one of claims 1 to 5.

52. siC5a1-M1SP, siC5b1-M1SP, siC5c1-M1SP, siC 5d1-M1SP, siC5e1-M1SP, or siC5f1-M1SP The siRNA according to any one of claims 31 to 51,

53. A composition comprising the siRNA according to any one of claims 31 to 52 and a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising:

54. siRNA according to any one of claims 31 to 52 and a compound complexed with said siRNA. The siRNA conjugate comprises a conjugated group.

55. siRNA complex according to any one of claims 1 to 30 and 54, or claims 31 to 52 The siRNA according to any one of claims 1 to 5 and / or the pharmaceutical composition according to claim 53 is administered to muscle tissue. Use in the preparation of a medicament for the treatment and / or prevention of asthenia.

56. A method for treating and / or preventing myasthenia gravis, comprising administering to a subject a compound according to any one of claims 1 to 30 and 54.

1. The siRNA complex according to any one of claims 31 to 52. and / or administering an effective amount of the pharmaceutical composition according to claim 53 to a subject suffering from myasthenia gravis. The method comprising administering.

57. A method for suppressing expression of C5 gene in hepatocytes, comprising any one of claims 1 to 30 and 54. The siRNA complex according to any one of claims 31 to 52, A method for treating hepatitis comprising contacting said hepatocytes with an effective amount of the pharmaceutical composition according to claim 53. Hmm, a method.

58. siRNA complex according to any one of claims 1 to 30 and 54, or claims 31 to 52 A kit comprising the siRNA according to any one of claims 53 and / or the pharmaceutical composition according to claim 53. to.

Images