RU2816898C2 - Nucleic acid, pharmaceutical composition and conjugate, method of production and use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a siRNA conjugate having a structure represented by formula (308), where L₁, M₁, R₂, R₃, R₁₀-R₁₅, m₁-m₃, n₁-n₃ are defined in the claims, and which siRNA is capable of inhibiting the expression of complement protein 5 (C5) gene, where each nucleotide in siRNA is an independent modified or unmodified nucleotide. siRNA contains a sense strand and an antisense strand. Sense strand contains a nucleotide sequence I, and the nucleotide sequence I has the same length and not more than three nucleotide differences from the nucleotide sequence shown in SEQ ID NO: 1. Antisense strand contains a nucleotide sequence II, and the nucleotide sequence II has the same length and not more than three nucleotide differences from the nucleotide sequence shown in SEQ ID NO: 2.

EFFECT: what is presented is using the siRNA conjugate for producing a drug for treating and / or preventing myasthenia gravis and a method for inhibiting expression of the C5 gene in hepatocyte.

24 cl, 2 dwg, 6 tbl, 4 ex

Description

TECHNICAL FIELD

The present invention relates to a nucleic acid, pharmaceutical composition and siRNA conjugate capable of inhibiting the expression of the complement protein 5 (C5) gene. The present invention also relates to a method for preparing and using said nucleic acid conjugate, pharmaceutical composition and siRNA conjugate.

BACKGROUND OF THE ART

Myasthenia gravis (MG) is an acquired autoimmune disease primarily mediated by acetylcholine receptor antibody (AchR-Ab), cellular immune dependence and complement involvement, and associated with the acetylcholine receptor (AChR) on the postsynaptic membrane of the neuromuscular junction.

Complement protein (C5) is one of the key targets for the treatment of myasthenia gravis. With the participation of complements, AChR-Ab combines with AChR, which leads to the destruction of large numbers of AChR through complement-mediated lysis of the cell membrane, resulting in muscle weakness due to disruption of acetylcholine signaling in the postsynaptic membrane. Research has shown that by specifically binding drugs to the complement protein C5, it is possible to prevent the cleavage of C5 into C5a and C5b, thereby preventing the formation of the membrane attack complex, blocking the damage to the neuromuscular junction by the membrane attack complex and the subsequent production of pro-inflammatory factors, thereby providing an immunosuppressive effect and treatment myasthenia gravis.

Small interfering RNA (siRNA), based on the RNA interference (RNAi) mechanism, can inhibit or block the expression of target genes of interest in a sequence-specific manner, thereby achieving the goal of treating diseases. If inhibition of C5 expression can be achieved to block the production of complement proteins, maintain normal immune functions and inhibit the pathological immune response at the mRNA level, this would undoubtedly represent the most advanced treatment option.

The key to developing siRNA-based drugs to inhibit C5 gene expression and treat myasthenia gravis is to find a suitable siRNA and modify it, as well as an effective delivery system.

BRIEF DESCRIPTION OF THE INVENTION

The present inventors have unexpectedly discovered that the siRNA conjugate of the present invention can specifically inhibit the expression of the C5 gene, specifically target the liver, inhibit the expression of the C5 gene in the liver, and treat or prevent myasthenia gravis. In addition, the inventors have also developed a highly active siRNA and pharmaceutical composition.

In some embodiments, the present invention provides an siRNA conjugate having the structure represented by Formula (308):

Formula (308),

where: n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4; each of m1, m2 and m3 independently represents an integer from 2 to 10; each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ or R ₁₅ independently represents H or is selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl and C ₁ -C ₁₀ alkoxy ;

R ₃ is a group having the structure represented by Formula A59:

(A59),

where E ₁ represents OH, SH or BH ₂ ; And

Nu is an siRNA; the specified siRNA contains a sense strand and an antisense strand, each nucleotide in the specified siRNA independently represents a modified or unmodified nucleotide, where the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II; nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region; and nucleotide sequence I and nucleotide sequence II are selected from the group of sequences according to the following i) to vi):

i) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 2 and differs from it by no more than three nucleotides:

5'-CUUCAUUCAUCAGACAAZ ₁ -3' (SEQ ID NO: 1);

5'-Z ₂ UUGUCUGUAUGAAUGAAG-3' (SEQ ID NO: 2),

where Z ₁ is A, Z ₂ is U, nucleotide sequence I contains nucleotide Z ₃ at the site corresponding to Z ₁ , nucleotide sequence II contains nucleotide Z ₄ at the site corresponding to Z ₂ , and Z ₄ is the first nucleotide with 5 '-end of the antisense strand;

ii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61, and differs from it by no more than three nucleotides; and nucleotide sequence II is the same length as the nucleotide sequence set forth in SEQ ID NO: 62 and differs from it by no more than three nucleotides:

5'-CUACAGUUUAGAAGAUUUZ ₅ -3' (SEQ ID NO: 61);

5'-Z ₆ AAAUCUUCUAAACUGUAG-3' (SEQ ID NO: 62),

where Z ₅ is A, Z ₆ is U, nucleotide sequence I contains nucleotide Z ₇ at the site corresponding to Z ₅ , nucleotide sequence II contains nucleotide Z ₈ at the site corresponding to Z ₆ , and Z ₈ represents the first nucleotide with 5 '-end of the antisense strand;

iii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 122 and differs from it by no more than three nucleotides:

5'-GGAAGGUUACCGAGCAAUZ ₉ -3' (SEQ ID NO: 121);

5'-Z ₁₀ AUUGCUCGGUAACCUUCC-3' (SEQ ID NO: 122),

where Z ₉ is A, Z ₁₀ is U, nucleotide sequence I contains nucleotide Z ₁₁ at the site corresponding to Z ₉ , nucleotide sequence II contains nucleotide Z ₁₂ at the site corresponding to Z ₁₀ , and Z ₁₂ represents the first nucleotide with 5 '-end of the antisense strand;

iv) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 182 and differs from it by no more than three nucleotides:

5'-AGAACAGACAGCAGAUUZ ₁₃ -3' (SEQ ID NO: 181);

5'-Z ₁₄ AAUUCUGCUGUCUGUUCU-3' (SEQ ID NO: 182),

where Z ₁₃ is A, Z ₁₄ is U, nucleotide sequence I contains nucleotide Z ₁₅ at the site corresponding to Z ₁₃ , nucleotide sequence II contains nucleotide Z ₁₆ at the site corresponding to Z ₁₄ , and Z ₁₆ represents the first nucleotide with 5 '-end of the antisense strand;

v) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 242 and differs from it by no more than three nucleotides:

5'-CCAAGAAGAACGCUGCAAZ ₁₇ -3' (SEQ ID NO: 241);

5'-Z ₁₈ UUGCAGCGUUCUUCUUGG-3' (SEQ ID NO: 242),

where Z ₁₇ is A, Z ₁₈ is U, nucleotide sequence I contains nucleotide Z ₁₉ at the site corresponding to Z ₁₇ , nucleotide sequence II contains nucleotide Z ₂₀ at the site corresponding to Z ₁₈ , and Z ₂₀ represents the first nucleotide with 5 '-end of the antisense strand; And

vi) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 301, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 302 and differs from it by no more than three nucleotides:

5'-CCAGUAAGCAAGCCAGAAZ ₂₁ -3' (SEQ ID NO: 301);

5'-Z ₂₂ UUCUGGCUUGCUUACUGG-3' (SEQ ID NO: 302),

where Z ₂₁ is A, Z ₂₂ is U, nucleotide sequence I contains nucleotide Z ₂₃ at the site corresponding to Z ₂₁ , nucleotide sequence II contains nucleotide Z ₂₄ at the site corresponding to Z ₂₂ , and Z ₂₄ is the first nucleotide with 5 '-end of the antisense strand;

R ₂ is a linear alkylene of 1 to 20 carbon atoms in length, where one or more carbon atoms are optionally substituted by any one or more of the group consisting of: C(O), NH, O, S, CH=N, S(O ) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene, and where R ₂ is optionally substituted by any one or more of the group , consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl-NH ₂ , -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -NH(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkylphenyl), -NH(C ₁ -C ₁₀ alkylphenyl), cyano, nitro, -CO ₂ H, -C(O)O( C ₁ -C ₁₀ alkyl), -CON(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ alkyl)C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)C( O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ - C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH(phenyl), -NHSO ₂ (C ₁ -C ₁₀ alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl);

each L ₁ is independently a linear alkylene of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally substituted by any one or more of the group consisting of: C(O), NH, O, S, CH=N, S (O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein L ₁ is optionally substituted by any one or more of the group consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl-NH ₂ , -N(C ₁ -C ₁₀ alkyl)(C ₁ - C ₁₀ alkyl), -NH(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkylphenyl), -NH(C ₁ -C ₁₀ alkylphenyl), cyano, nitro, -CO ₂ H, -C(O)O(C ₁ -C ₁₀ alkyl), -CON(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ alkyl)C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)C(O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ - C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH (phenyl), -NHSO ₂ (C ₁ -C ₁₀ alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C ₁ - C ₁₀ haloalkyl); And

denotes the site of covalent attachment of a group; and M ₁ represents a targeting group.

In some embodiments, the present invention provides an siRNA capable of inhibiting the expression of a C5 gene, wherein the siRNA comprises a sense strand and an antisense strand, and each nucleotide in the sense strand and antisense strand is independently a fluorine-modified nucleotide or a nucleotide with a non-fluorine modification; the sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region, fluorine-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, in the direction from 5'-end to 3'-end, nucleotides at positions 7, 8 and 9 of nucleotide sequence I in the sense strand are fluorine-modified nucleotides, and nucleotides at the remaining positions in the sense strand are non-fluorine-modified nucleotides; in a 5' to 3' direction, nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence II in the antisense strand are fluorine-modified nucleotides, and nucleotides at the remaining positions in the antisense strand are non-fluorine-modified nucleotides , and the nucleotide sequence I and the nucleotide sequence II are selected from one of the above i) to vi).

In some embodiments, the present invention provides a pharmaceutical composition comprising the aforementioned siRNA of the present invention and a pharmaceutically acceptable carrier.

In some embodiments, the present invention provides an siRNA conjugate comprising the aforementioned siRNA of the present invention and a conjugating group linked by conjugation to said siRNA.

Some embodiments of the present invention provide the use of an siRNA and/or a pharmaceutical composition and/or a siRNA conjugate of the present invention for the production of a medicament for the treatment and/or prevention of myasthenia gravis.

In some embodiments, the present invention provides a method of treating and/or preventing myasthenia gravis, comprising administering an effective amount of siRNA and/or a pharmaceutical composition and/or siRNA conjugate of the present invention to a subject suffering from myasthenia gravis.

In some embodiments, the present invention provides a method of inhibiting C5 gene expression in a hepatocyte, comprising bringing an effective amount of siRNA and/or a pharmaceutical composition and/or siRNA conjugate of the present invention into contact with said hepatocyte.

In some embodiments, the present invention provides a kit containing siRNA and/or a pharmaceutical composition and/or siRNA conjugate according to the present invention.

Positive effects

In some embodiments, the siRNA, pharmaceutical composition and siRNA conjugate of the present invention have better stability, higher C5 mRNA inhibitory activity and lower off-target effects, and/or can significantly treat or alleviate the symptoms of myasthenia gravis.

In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits superior inhibitory activity against target mRNA in in vitro cell experiments. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage of inhibition of the target mRNA in a hepatocyte of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

In some embodiments, the siRNA of the present invention exhibits greater inhibitory activity in HepG2 cells, and the IC ₅₀ for C5 mRNA ranges from 1.494 nM to 9.688 nM. In some embodiments, a fluorescently tagged siRNA conjugate of the present invention is injected subcutaneously into C57 mice to perform real-time fluorescence imaging on the mice and monitor the distribution of fluorescence across organs. After 48 hours, the mice were sacrificed for organ dissection, and it was found that almost all of the siRNA conjugates accumulated in the liver, indicating that the siRNA conjugate of the present invention can efficiently and specifically deliver siRNA to the liver, indicating that the conjugate can specifically inhibit the expression of target mRNA in the liver.

In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention may exhibit greater stability and/or greater activity in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage of target mRNA inhibition of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95 %, in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage inhibition of C5 mRNA expression equal to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95 %, in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage inhibition of hepatic C5 mRNA expression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage inhibition of C5 mRNA expression in the liver of model animals of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention exhibits a percentage inhibition of hepatic C5 mRNA expression in humans of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, in vivo.

In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate of the present invention does not exhibit significant off-target effects. An off-target effect may be, for example, inhibition of normal expression of a gene that is not the target gene. It is considered non-significant if the binding/inhibition of non-target gene expression is below 50%, 40%, 30%, 20% or 10% of the target effect.

It follows that the siRNA, pharmaceutical composition and siRNA conjugate of the present invention can inhibit the expression of C5 mRNA, effectively treat and/or prevent the symptoms of myasthenia gravis, and have good application prospects.

Other features and advantages of the present invention will be described in detail in the following section of the detailed description of the invention.

DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1A-1H are dose-response curves fitted based on the relative levels of C5 mRNA expression in HepG2 cells following transfection of various conjugates 1-8.

FIG. 2A is a photograph of fluorescence imaging of various organs in C57 mice 48 hours after administration of 5 ml/kg 1× PBS, 3 mg/kg Cy5-siRNA 1, or 3 mg/kg Cy5-conjugate 1.

FIG. 2B is a photograph of fluorescence imaging of various organs in C57 mice 48 hours after administration of 5 ml/kg 1× PBS, 3 mg/kg Cy5-siRNA 2, or 3 mg/kg Cy5-conjugate 2.

DETAILED DESCRIPTION OF THE INVENTION

Specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein serve only to illustrate and explain the present invention and do not serve to limit the present invention in any respect.

In the present invention, C5 mRNA refers to the mRNA with the sequence presented under Genbank accession number M57729.1. In addition, unless otherwise specified, the term “target gene” as used in the present invention refers to a gene capable of transcribing the above C5 mRNA, and the term “target mRNA” refers to the above C5 mRNA.

Definitions

In the context of the present invention, unless otherwise indicated, the capital letters C, G, U and A indicate the bases of the nucleotides; a lowercase m indicates that the nucleotide to the left of the m is a methoxy-modified nucleotide; a lowercase f indicates that the nucleotide to the left of the f is a fluorine-modified nucleotide; the lowercase s indicates that the two nucleotides to the left and right of the s are joined by a thiophosphate; P1 indicates that the nucleotide to the right of P1 is a nucleotide with a 5'-phosphate group or a nucleotide modified with a 5'-phosphate analogue, the letter combination VP indicates that the nucleotide to the right of the letter combination VP is a vinyl phosphate modified nucleotide, the letter combination Ps indicates that the nucleotide located to the right of the letter combination Ps is a thiophosphate-modified nucleotide, and the capital letter P indicates that the nucleotide located to the right of the letter P is a nucleotide with a 5'-phosphate group.

In the context of the present invention, a "fluorine-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxy group of a ribose group of a nucleotide with fluorine, and the term "non-fluorine modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxy group of a ribose group of a nucleotide with a , other than fluorine, or a nucleotide analogue. "Nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid and is structurally different from an adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymidine deoxyribonucleotide, such as an isonucleotide, a bridging nucleic acid (BNA) nucleotide or acyclic nucleotide. "Methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxy group of a ribose group with a methoxy group.

In the context of the present invention, the expressions “complementary” and “reverse complementary” can be used interchangeably and have the meaning generally known in the art, namely, bases on one strand are complementarily paired with bases on the other strand of a double-stranded nucleic acid molecule. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); and the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair contains a purine and a pyrimidine. If adenines on one chain are always paired with thymines (or uracils) on the other chain, and guanines are always paired with cytosines, the two chains are considered complementary to each other; and the sequence of a strand can be inferred from the sequence of its complementary strand. Accordingly, “mispairing” means that in a double-stranded nucleic acid the bases at the corresponding sites are not represented as complementary pairs.

In the context of the present invention, unless otherwise indicated, “predominantly reverse complementary” means that no more than 3 mismatched bases are present between two nucleotide sequences. “Substantially reverse complementary” means that there is no more than 1 mismatched base between two nucleotide sequences. “Fully complementary” means that there are no mismatched bases between two nucleotide sequences.

In the context of the present invention, if a nucleotide sequence has a "nucleotide difference" from another nucleotide sequence, the nucleotide bases at the same position therein are changed. For example, if the nucleotide base in the second sequence is A, and the nucleotide base at the same position in the first sequence is U, C, G, or T, the two nucleotide sequences are considered to have a difference in the nucleotide at that position. In some embodiments, if a nucleotide at any position is replaced by a baseless nucleotide or nucleotide analogue, there is also a nucleotide difference at that position.

In the context of the present invention, in particular, in the description of the method for producing siRNA, a composition containing siRNA, or a siRNA conjugate according to the present invention, unless otherwise indicated, the nucleoside monomer refers, in accordance with the type and sequence of nucleotides in the siRNA or siRNA conjugate that is required obtain, to unmodified or modified ribonucleoside amidophosphites used in solid-phase amidophosphite synthesis (RNA amidophosphites are also called nucleoside amidophosphites elsewhere). Solid phase amidophosphite synthesis is a method well known to those skilled in the art and used in RNA synthesis. Nucleoside monomers used in the present invention may be commercially available.

In the context of the present invention, unless otherwise indicated, "conjugation" refers to two or more chemical moieties, each having a specific function, connected to each other by a covalent bond. Accordingly, “conjugate” refers to a compound formed by the covalent linkage of individual chemical moieties. Furthermore, a “siRNA conjugate” is a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA. Hereinafter, the siRNA conjugate of the present invention is sometimes abbreviated as “conjugate.” siRNA conjugate should be understood according to the context as a general term for a variety of siRNA conjugates or siRNA conjugates represented by specific chemical formulas. In the context of the present invention, the term "conjugating molecule" should be understood as a specific compound that can be conjugated to siRNA through reactions to ultimately obtain the siRNA conjugate of the present invention.

As used herein, the term “optional” or “optional” means that the event or condition described below may or may not occur, and that the description includes instances in which the event or condition may or may not occur. For example, the term "optionally substituted" "alkyl" includes both "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will recognize, with respect to any group containing one or more substituents, that such groups do not involve the introduction of any substitution or type of substitution that is sterically unfavorable, synthetically infeasible and/or unstable in nature.

As used herein, "alkyl" refers to straight chain and branched chain having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g. 1 to 10 carbon atoms, e.g. 1 to 8 or 1 to 6 atoms carbon. For example, C ₁ -C ₆ alkyl includes both straight and branched chain alkyl having from 1 to 6 carbon atoms. When referring to an alkyl moiety having a specific number of carbon atoms, it is intended to include all branched chain and straight chain forms having that number of carbon atoms; thus, for example, "butyl" means n-butyl, sec-butyl, isobutyl and tert-butyl; and by “propyl” we mean n-propyl and isopropyl. Alkylene is a species of alkyl, belonging to the same radicals as alkyl, but having two attachment positions.

As used herein, “alkenyl” refers to an unsaturated branched or straight alkyl having at least one carbon-carbon double bond formed by the corresponding removal of one hydrogen molecule from two adjacent carbon atoms of the parent alkyl. The group may have a cis or trans double bond configuration. Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl) and prop-2-en-2-yl; and butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2 -en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl and the like. In some embodiments, the alkenyl group has from 2 to 20 carbon atoms, and in other embodiments from 2 to 10, from 2 to 8, or from 2 to 6 carbon atoms. Alkenylene is a species of alkenyl belonging to the same radicals as alkenyl but having two attachment positions.

As used herein, “alkynyl” refers to an unsaturated branched or straight alkyl having at least one carbon-carbon triple bond formed by the corresponding removal of two hydrogen molecules from two adjacent carbon atoms of the parent alkyl. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-1-yl and prop-2-yn-1-yl; and butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like. In some embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a species of alkynyl, belonging to the same residues as alkynyl, but having two attachment positions.

As used herein, "alkoxy" refers to an alkyl group of a specified number of carbon atoms, attached via an oxygen bridge, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy and the like. Alkoxy typically has from 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached via an oxygen bridge.

As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a carbon atom of the ring. An aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and from 6 to 18 carbon atoms, and at least one ring in the ring system is completely unsaturated, i.e. contains a cyclic delocalized (4n+2)π-electron system in accordance with Hückel's theory. Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl and the like. Arylene is a species of aryl that has the same moieties as aryl but has two attachment positions.

As used herein, "halogen substituent" or "halogen" refers to fluorine, chlorine, bromine and iodine, and the term "halogen" includes fluorine, chlorine, bromine or iodine.

As used herein, “haloalkyl” refers to an alkyl as defined above in which a specified number of carbon atoms is replaced by one or more halogen atoms, up to the maximum number of halogen atoms allowed. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.

"Heterocyclyl" refers to a stable 3-18 membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen or sulfur. Unless otherwise indicated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may contain fused or bridged ring systems. The heteroatoms in the heterocyclyl may optionally be oxidized. One or more nitrogen atoms, if present, are not necessarily quaternized. Heterocyclyl is partially or fully saturated. The heterocyclyl can be connected to the rest of the molecule through any ring atom. Examples of such heterocyclyl include, but are not limited to, dioxanyl, thienyl[1,3]disulfonyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyr rolidinyl, oxazolidinyl, piperidinyl , piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl and 1,1-dioxothiomorpholinyl.

"Heteroaryl" refers to a group derived from a 3 to 18 membered aromatic ring radical containing from 2 to 17 carbon atoms and from 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. For purposes of the present invention, heteroaryl may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is completely unsaturated, i.e., contains a cyclic delocalized (4n+2)π-electron system in accordance with Hückel's theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl are optionally oxidized. One or more nitrogen atoms, if present, are not necessarily quaternized. Heteroaryl can be connected to the rest of the molecule through any ring atom. Examples of such heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzidolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b] [1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1 ,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]-thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h] quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo[3,2- c]pyridinyl, 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]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methane-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6- naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H- cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3- d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl and thiophenyl/thienyl.

Various hydroxy protecting groups can be used in the present invention. In general, protecting groups render chemical functional groups inert to certain reaction conditions and can be attached to and removed from such functional groups in a molecule without significant damage to the rest of the molecule. Exemplary hydroxy protecting groups are disclosed in Tetrahedron 1992, 48, 2223-2311 per ed. Beaucage, et al., and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under basic conditions but may be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), or 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

As used herein, the term "subject" refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, human, non-human primate (eg, rhesus or other macaque species), mouse, pig, horse, donkey, cow, sheep, rat, and any species of poultry.

As used herein, “treatment” refers to a method of obtaining a beneficial or desired result, including, but not limited to, a therapeutic effect. "Therapeutic effect" means the elimination or improvement of the potential disorder being treated. Moreover, a therapeutic effect is achieved by eliminating or reducing one or more physiological symptoms associated with the potential disorder such that the subject shows improvement although the subject may still be affected by the potential disorder.

As used herein, “prevention” refers to a method of obtaining a beneficial or intended result, including, but not limited to, a prophylactic effect. To achieve a “prophylactic effect,” the siRNA conjugate or composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

siRNA

In one aspect, the present invention provides six types of siRNAs capable of inhibiting C5 gene expression.

The siRNA of the present invention contains nucleotides as basic structural units. It is well known to those skilled in the art that a nucleotide contains a phosphate group, a ribose group and a base. Detailed illustrations relating to such groups are not presented in this document.

The siRNA of the present invention contains a sense strand and an antisense strand, wherein the lengths of the sense strand and the antisense strand are the same or different, the length of the sense strand is 19-23 nucleotides, and the length of the antisense strand is 19-26 nucleotides. Thus, the ratio of the sense strand length to the antisense strand length of the siRNA according to the present invention can be 19/19, 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20 /20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25 , 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23 /24, 23/25 or 23/26. In some embodiments, the ratio of the sense strand length to the antisense strand length of the siRNA is 19/21, 21/23, or 23/25.

First siRNA

According to the present invention, the siRNA may be a first siRNA.

The first siRNA contains a sense strand and an antisense strand. Each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region where the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 2 and differs from it by no more than three nucleotides:

5'-CUUCAUUCAUCAGACAAZ ₁ -3' (SEQ ID NO: 1);

5'-Z ₂ UUGUCUGUAUGAAUGAAG-3' (SEQ ID NO: 2),

where Z ₁ is A, Z ₂ is U, nucleotide sequence I contains nucleotide Z ₃ at the site corresponding to Z ₁ , nucleotide sequence II contains nucleotide Z ₄ at the site corresponding to Z ₂ , and Z ₄ is the first nucleotide with 5 '-end of the antisense strand.

As used herein, the term "corresponding site" means being at the same site in a nucleotide sequence when starting from the same end of the nucleotide sequence. For example, the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide at the site corresponding to the first nucleotide at the 3' end of SEQ ID NO: 1.

In some embodiments, the sense strand contains exclusively nucleotide sequence I, and the antisense strand contains exclusively nucleotide sequence II.

In some embodiments, nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 1 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 2 by no more than one nucleotide.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 2 includes a difference in the Z ₄ site, and Z ₄ is selected from A, C, or G. In some embodiments, the nucleotide difference is the difference is the Z ₄ site, and Z ₄ is selected from A, C, or G. In some embodiments, Z ₃ is a nucleotide complementary to Z ₄ . siRNAs having the above nucleotide difference have a higher ability of the siRNA to inhibit the target mRNA, and these siRNAs are also included in the scope of the present invention.

In some embodiments, nucleotide sequence I is substantially reverse complementary, substantially reverse complementary, or completely reverse complementary to nucleotide sequence II. “Mostly reverse complementary” means the presence of no more than three mismatched bases between two nucleotide sequences; “substantially reverse complementary” means the presence of no more than one mispaired base between two nucleotide sequences; and “fully reverse complementary” means that there is no mispaired base between two nucleotide sequences.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 3, and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 4:

5'-CUUCAUUCAUCAGACAAZ ₃ -3' (SEQ ID NO: 3);

5'-Z ₄ UUGUCUGUAUGAAUGAAG-3' (SEQ ID NO: 4),

where Z ₄ represents the first nucleotide from the 5' end of the antisense strand; Z ₃ selected from A, U, G or C; and Z ₄ is a nucleotide complementary to Z ₃ ; and in some embodiments, Z ₃ is A and Z ₄ is U.

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, and nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III has the same length and is substantially reverse complementary or completely reverse complementary to nucleotide sequence IV; nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. In some embodiments, nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, and nucleotide sequence II refers to a nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 1 in the target mRNA, and having the same length as the nucleotide sequence IV.

In some embodiments, nucleotide sequence III and nucleotide sequence IV are both one nucleotide in length. The base of nucleotide sequence III is U, and the base of nucleotide sequence IV is A; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 20/20; or nucleotide sequences III and IV are both two nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CU and the base composition of nucleotide sequence IV is AG; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 21/21; or nucleotide sequences III and IV are both three nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UCU and the base composition of nucleotide sequence IV is AGA; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 22/22; or nucleotide sequences III and IV are both four nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is UUCU and the base composition of nucleotide sequence IV is AGAA; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23/23. In some embodiments, nucleotide sequence III and nucleotide sequence IV are two nucleotides in length, and in the 5' to 3' end direction, the base composition of nucleotide sequence III is CU and the base composition of nucleotide sequence IV is AG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21/21.

In some embodiments, nucleotide sequence III is completely reverse complementary to nucleotide sequence IV. Thus, if the base(s) of nucleotide sequence III is known, then the base(s) of nucleotide sequence IV can also be determined.

Second miRNA

According to the present invention, the siRNA may be a second siRNA.

The second siRNA contains a sense strand and an antisense strand. Each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region where the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61, and differs from it by no more than three nucleotides; and nucleotide sequence II is the same length as the nucleotide sequence set forth in SEQ ID NO: 62 and differs from it by no more than three nucleotides:

5'-CUACAGUUUAGAAGAUUUZ ₅ -3' (SEQ ID NO: 61);

5'-Z ₆ AAAUCUUCUAAACUGUAG-3' (SEQ ID NO: 62),

where Z ₅ is A, Z ₆ is U, nucleotide sequence I contains nucleotide Z ₇ at the site corresponding to Z ₅ , nucleotide sequence II contains nucleotide Z ₈ at the site corresponding to Z ₆ , and Z ₈ represents the first nucleotide with 5 '-end of the antisense strand.

In some embodiments, the sense strand contains exclusively nucleotide sequence I, and the antisense strand contains exclusively nucleotide sequence II.

In some embodiments, nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 61 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 62 by no more than one nucleotide.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 62 includes a difference in site Z ₈ , and Z ₈ is selected from A, C, or G. In some embodiments, the nucleotide difference is the difference is the Z ₈ site, and Z ₈ is selected from A, C, or G. In some embodiments, Z ₇ is a nucleotide complementary to Z ₈ . siRNAs having the above nucleotide difference have a higher ability of the siRNA to inhibit the target mRNA, and these siRNAs are also included in the scope of the present invention.

In some embodiments, nucleotide sequence I is substantially reverse complementary, substantially reverse complementary, or completely reverse complementary to nucleotide sequence II.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 63, and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 64:

5'-CUACAGUUUAGAAGAUUUZ ₇ -3' (SEQ ID NO: 63);

5'-Z ₈ AAAUCUUCUAAACUGUAG-3' (SEQ ID NO: 64),

where Z ₈ represents the first nucleotide from the 5' end of the antisense strand; Z ₇ selected from A, U, G or C; and Z ₈ is a nucleotide complementary to Z ₇ ; and in some embodiments, Z ₇ is A and Z ₈ is U.

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, and nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III has the same length and is substantially reverse complementary or completely reverse complementary to nucleotide sequence IV; nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, and nucleotide sequence II refers to the nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 61 in the target mRNA and having the same length , the same as the nucleotide sequence of IV.

In some embodiments, nucleotide sequence III and nucleotide sequence IV are both one nucleotide in length. The base of nucleotide sequence III is A, and the base of nucleotide sequence IV is U; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 20/20; or nucleotide sequences III and IV are both two nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UA, and the base composition of nucleotide sequence IV is UA; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 21/21; or nucleotide sequences III and IV are both three nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is AUA and the base composition of nucleotide sequence IV is UAU; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 22/22; or nucleotide sequences III and IV are both four nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CAUA and the base composition of nucleotide sequence IV is UAUG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23/23. In some embodiments, nucleotide sequence III and nucleotide sequence IV are two nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UA, and the base composition of nucleotide sequence IV is UA; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21/21.

In some embodiments, nucleotide sequence III is completely reverse complementary to nucleotide sequence IV. Thus, if the base(s) of nucleotide sequence III is known, then the base(s) of nucleotide sequence IV can also be determined.

Third miRNA

According to the present invention, the siRNA may be a third siRNA.

The third siRNA contains a sense strand and an antisense strand. Each nucleotide in the third siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region where the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 122 and differs from it by no more than three nucleotides:

5'-GGAAGGUUACCGAGCAAUZ ₉ -3' (SEQ ID NO: 121);

5'-Z ₁₀ AUUGCUCGGUAACCUUCC-3' (SEQ ID NO: 122),

where Z ₉ is A, Z ₁₀ is U, nucleotide sequence I contains nucleotide Z ₁₁ at the site corresponding to Z ₉ , nucleotide sequence II contains nucleotide Z ₁₂ at the site corresponding to Z ₁₀ , and Z ₁₂ represents the first nucleotide with 5 '-end of the antisense strand.

In some embodiments, the sense strand contains exclusively nucleotide sequence I, and the antisense strand contains exclusively nucleotide sequence II.

In some embodiments, nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 121 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 122 by no more than one nucleotide.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 122 includes a difference in site Z ₁₂ , and Z ₁₂ is selected from A, C, or G. In some embodiments, the nucleotide difference is the difference is the Z ₁₂ site, and Z ₁₂ is selected from A, C, or G. In some embodiments, Z ₁₁ is a nucleotide complementary to Z ₁₂ . siRNAs having the above nucleotide difference have a higher ability of the siRNA to inhibit the target mRNA, and these siRNAs are also included in the scope of the present invention.

In some embodiments, nucleotide sequence I is substantially reverse complementary, substantially reverse complementary, or completely reverse complementary to nucleotide sequence II.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 123, and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 124:

5'-GGAAGGUUACCGAGCAAUZ ₁₁ -3' (SEQ ID NO: 123);

5'-Z ₁₂ AUUGCUCGGUAACCUUCC-3' (SEQ ID NO: 124),

where Z ₁₂ represents the first nucleotide from the 5' end of the antisense strand; Z ₁₁ selected from A, U, G or C; and Z ₁₂ is a nucleotide complementary to Z ₁₁ ; and in some embodiments, Z ₁₁ is A and Z ₁₂ is U.

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, and nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III has the same length and is substantially reverse complementary or completely reverse complementary to nucleotide sequence IV; nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, and nucleotide sequence II refers to the nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 121 in the target mRNA and having the same length , the same as the nucleotide sequence of IV.

In some embodiments, nucleotide sequence III and nucleotide sequence IV are both one nucleotide in length. The base of nucleotide sequence III is G and the base of nucleotide sequence IV is C; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 20/20; or nucleotide sequences III and IV are both two nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is AG, and the base composition of nucleotide sequence IV is CU; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 21/21; or nucleotide sequences III and IV are both three nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CAG and the base composition of nucleotide sequence IV is CUG; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 22/22; or nucleotide sequences III and IV are both four nucleotides in length, and in the 5' to 3' direction, the base composition of nucleotide sequence III is CCAG and the base composition of nucleotide sequence IV is CUGG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23/23. In some embodiments, nucleotide sequence III and nucleotide sequence IV are two nucleotides in length, and in the 5' to 3' end direction, the base composition of nucleotide sequence III is AG and the base composition of nucleotide sequence IV is CU; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21/21.

In some embodiments, nucleotide sequence III is completely reverse complementary to nucleotide sequence IV. Thus, if the base(s) of nucleotide sequence III is known, then the base(s) of nucleotide sequence IV can also be determined.

Fourth miRNA

According to the present invention, the siRNA may be a fourth siRNA.

The fourth siRNA contains a sense strand and an antisense strand. Each nucleotide in the fourth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region where the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 182 and differs from it by no more than three nucleotides:

5'-AGAACAGACAGCAGAUUZ ₁₃ -3' (SEQ ID NO: 181);

5'-Z ₁₄ AAUUCUGCUGUCUGUUCU-3' (SEQ ID NO: 182),

where Z ₁₃ is A, Z ₁₄ is U, nucleotide sequence I contains nucleotide Z ₁₅ at the site corresponding to Z ₁₃ , nucleotide sequence II contains nucleotide Z ₁₆ at the site corresponding to Z ₁₄ , and Z ₁₆ represents the first nucleotide with 5 '-end of the antisense strand.

In some embodiments, the sense strand contains exclusively nucleotide sequence I, and the antisense strand contains exclusively nucleotide sequence II.

In some embodiments, nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 181 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 182 by no more than one nucleotide.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 182 includes a difference in the Z ₁₆ site, and Z ₁₆ is selected from A, C, or G. In some embodiments, the nucleotide difference is the difference is the Z ₁₆ site, and Z ₁₆ is selected from A, C, or G. In some embodiments, Z ₁₅ is a nucleotide complementary to Z ₁₆ . siRNAs having the above nucleotide difference have a higher ability of the siRNA to inhibit the target mRNA, and these siRNAs are also included in the scope of the present invention.

In some embodiments, nucleotide sequence I is substantially reverse complementary, substantially reverse complementary, or completely reverse complementary to nucleotide sequence II.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 183, and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 184:

5'-AGAACAGACAGCAGAUUZ ₁₅ -3' (SEQ ID NO: 183);

5'-Z ₁₆ AAUUCUGCUGUCUGUUCU-3' (SEQ ID NO: 184),

where Z ₁₆ represents the first nucleotide from the 5' end of the antisense strand; Z ₁₅ selected from A, U, G or C; and Z ₁₆ is a nucleotide complementary to Z ₁₅ ; and in some embodiments, Z ₁₅ is A and Z ₁₆ is U.

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, and nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III has the same length and is substantially reverse complementary or completely reverse complementary to nucleotide sequence IV; nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, and nucleotide sequence II refers to the nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 181 in the target mRNA and having the same length , the same as the nucleotide sequence of IV.

In some embodiments, nucleotide sequence III and nucleotide sequence IV are both one nucleotide in length. The base of nucleotide sequence III is G and the base of nucleotide sequence IV is C; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 20/20; or nucleotide sequences III and IV are both two nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is GG and the base composition of nucleotide sequence IV is CC; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 21/21; or nucleotide sequences III and IV are both three nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is AGG and the base composition of nucleotide sequence IV is CCU; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 22/22; or nucleotide sequences III and IV are both four nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CAGG and the base composition of nucleotide sequence IV is CCUG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23/23. In some embodiments, nucleotide sequence III and nucleotide sequence IV are two nucleotides in length, and in the 5' to 3' end direction, the base composition of nucleotide sequence III is GG and the base composition of nucleotide sequence IV is CC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21/21.

In some embodiments, nucleotide sequence III is completely reverse complementary to nucleotide sequence IV. Thus, if the base(s) of nucleotide sequence III is known, then the base(s) of nucleotide sequence IV can also be determined.

Fifth miRNA

According to the present invention, the siRNA may be a fifth siRNA.

The fifth miRNA contains a sense strand and an antisense strand. Each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region where the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 242 and differs from it by no more than three nucleotides:

5'-CCAAGAAGAACGCUGCAAZ ₁₇ -3' (SEQ ID NO: 241);

5'-Z ₁₈ UUGCAGCGUUCUUCUUGG-3' (SEQ ID NO: 242),

where Z ₁₇ is A, Z ₁₈ is U, nucleotide sequence I contains nucleotide Z ₁₉ at the site corresponding to Z ₁₇ , nucleotide sequence II contains nucleotide Z ₂₀ at the site corresponding to Z ₁₈ , and Z ₂₀ represents the first nucleotide with 5 '-end of the antisense strand.

In some embodiments, the sense strand contains exclusively nucleotide sequence I, and the antisense strand contains exclusively nucleotide sequence II.

In some embodiments, nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 241 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 242 by no more than one nucleotide.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence set forth in SEQ ID NO: 242 includes a difference in the Z ₂₀ site, and Z ₂₀ is selected from A, C, or G. In some embodiments, the nucleotide difference is the difference is the Z ₂₀ site, and Z ₂₀ is selected from A, C, or G. In some embodiments, Z ₁₉ is a nucleotide complementary to Z ₂₀ . siRNAs having the above nucleotide difference have a higher ability of the siRNA to inhibit the target mRNA, and these siRNAs are also included in the scope of the present invention.

In some embodiments, nucleotide sequence I is substantially reverse complementary, substantially reverse complementary, or completely reverse complementary to nucleotide sequence II.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 243, and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 244:

5'-CCAAGAAGAACGCUGCAAZ ₁₉ -3' (SEQ ID NO: 243);

5'-Z ₂₀ UUGCAGCGUUCUUCUUGG-3' (SEQ ID NO: 244),

where Z ₂₀ represents the first nucleotide from the 5' end of the antisense strand; Z ₁₉ selected from A, U, G or C; and Z ₂₀ is a nucleotide complementary to Z ₁₉ ; and in some embodiments, Z ₁₉ is A and Z ₂₀ is U.

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, and nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III has the same length and is substantially reverse complementary or completely reverse complementary to nucleotide sequence IV; nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, and nucleotide sequence II refers to the nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 241 in the target mRNA and having the same length , the same as the nucleotide sequence of IV.

In some embodiments, nucleotide sequence III and nucleotide sequence IV are both one nucleotide in length. The base of nucleotide sequence III is G and the base of nucleotide sequence IV is C; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 20/20; or nucleotide sequences III and IV are both two nucleotides in length, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is GG and the base composition of nucleotide sequence IV is CC; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 21/21; or nucleotide sequences III and IV are both three nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is AGG and the base composition of nucleotide sequence IV is CCU; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 22/22; or nucleotide sequences III and IV are both four nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CAGG and the base composition of nucleotide sequence IV is CCUG; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23/23. In some embodiments, nucleotide sequence III and nucleotide sequence IV are two nucleotides in length, and in the 5' to 3' end direction, the base composition of nucleotide sequence III is GG and the base composition of nucleotide sequence IV is CC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21/21.

In some embodiments, nucleotide sequence III is completely reverse complementary to nucleotide sequence IV. Thus, if the base(s) of nucleotide sequence III is known, then the base(s) of nucleotide sequence IV can also be determined.

Sixth miRNA

According to the present invention, the siRNA may be a sixth siRNA.

The sixth miRNA contains a sense strand and an antisense strand. Each nucleotide in the sixth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region where the nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 301, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 302 and differs from it by no more than three nucleotides:

5'-CCAGUAAGCAAGCCAGAAZ ₂₁ -3' (SEQ ID NO: 301);

5'-Z ₂₂ UUCUGGCUUGCUUACUGG-3' (SEQ ID NO: 302),

where Z ₂₁ is A, Z ₂₂ is U, nucleotide sequence I contains nucleotide Z ₂₃ at the site corresponding to Z ₂₁ , nucleotide sequence II contains nucleotide Z ₂₄ at the site corresponding to Z ₂₂ , and Z ₂₄ is the first nucleotide with 5 '-end of the antisense strand.

In some embodiments, the sense strand contains exclusively nucleotide sequence I, and the antisense strand contains exclusively nucleotide sequence II.

In some embodiments, nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 301 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 302 by no more than one nucleotide.

In some embodiments, the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 302 includes a difference in the Z ₂₄ site, and Z ₂₄ is selected from A, C, or G. In some embodiments, the nucleotide difference is the difference is the Z ₂₄ site, and Z ₂₄ is selected from A, C, or G. In some embodiments, Z ₂₃ is a nucleotide complementary to Z ₂₄ . siRNAs having the above nucleotide difference have a higher ability of the siRNA to inhibit the target mRNA, and these siRNAs are also included in the scope of the present invention.

In some embodiments, nucleotide sequence I is substantially reverse complementary, substantially reverse complementary, or completely reverse complementary to nucleotide sequence II.

In some embodiments, nucleotide sequence I is the nucleotide sequence set forth in SEQ ID NO: 303, and nucleotide sequence II is the nucleotide sequence set forth in SEQ ID NO: 304:

5'-CCAGUAAGCAAGCCAGAAZ ₂₃ -3' (SEQ ID NO: 303);

5'-Z ₂₄ UUCUGGCUUGCUUACUGG-3' (SEQ ID NO: 304),

where Z ₂₄ represents the first nucleotide from the 5' end of the antisense strand; Z ₂₃ selected from A, U, G or C; and Z ₂₄ is a nucleotide complementary to Z ₂₃ ; and in some embodiments, Z ₂₃ is A and Z ₂₄ is U.

In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, and nucleotide sequence III and nucleotide sequence IV are each independently 1-4 nucleotides in length; nucleotide sequence III has the same length and is substantially reverse complementary or completely reverse complementary to nucleotide sequence IV; nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, and nucleotide sequence II refers to the nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 301 in the target mRNA and having the same length , the same as the nucleotide sequence of IV.

In some embodiments, nucleotide sequence III and nucleotide sequence IV are both one nucleotide in length. The base of nucleotide sequence III is A, and the base of nucleotide sequence IV is U; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 20/20; or nucleotide sequences III and IV are both two nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UA, and the base composition of nucleotide sequence IV is UA; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 21/21; or nucleotide sequences III and IV are both three nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UUA and the base composition of nucleotide sequence IV is UAA; in this case, the ratio of the length of the sense chain to the length of the antisense chain is 22/22; or nucleotide sequences III and IV are both four nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is GUUA and the base composition of nucleotide sequence IV is UAAC; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 23/23. In some embodiments, nucleotide sequence III and nucleotide sequence IV are two nucleotides in length, and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UA, and the base composition of nucleotide sequence IV is UA; in this case, the ratio of the length of the sense strand to the length of the antisense strand is 21/21.

In some embodiments, nucleotide sequence III is completely reverse complementary to nucleotide sequence IV. Thus, if the base(s) of nucleotide sequence III is known, then the base(s) of nucleotide sequence IV can also be determined.

Protruding ends and siRNA modification

The following description of the V nucleotide sequence, the nucleic acid sequence, the nucleotide modification in the miRNA and the modified sequence applies to any of the first to sixth miRNAs. That is, unless otherwise indicated, the following description of the miRNA should be considered to describe the first miRNA, second miRNA, third miRNA, fourth miRNA, fifth miRNA, and sixth miRNA in turn. For example, if a specific miRNA is not specified, "miRNA further comprises a V nucleotide sequence" means "a first miRNA, a second miRNA, a third miRNA, a fourth miRNA, a fifth miRNA, or a sixth miRNA further comprises a V nucleotide sequence."

In some embodiments, the sense strand and the antisense strand are of different lengths. Nucleotide sequence II further contains a nucleotide sequence V, which is 1-3 nucleotides in length and is linked to the 3' end of the antisense strand to form a 3' overhanging end of the antisense strand. Thus, the ratio of the length of the sense strand to the length of the antisense strand in the siRNA according to the present invention can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26. In some embodiments, the V nucleotide sequence is 2 nucleotides in length. Thus, the ratio of the length of the sense strand to the length of the antisense strand in the siRNA according to the present invention may be 19/21, 21/23 or 23/25.

Each nucleotide in the V nucleotide sequence may be any nucleotide. To simplify the synthesis and reduce the cost of its implementation, the nucleotide sequence V is 2 thymidine deoxyribonucleotides in a row (dTdT) or 2 uracil ribonucleotides in a row (UU); or, to improve the affinity of the antisense strand of the siRNA for the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding site of the target mRNA. Therefore, in some embodiments, the ratio of sense strand length to antisense strand length of the siRNA of the present invention is 19/21 or 21/23. In this case, the siRNA according to the present invention has better silencing activity against mRNA.

A nucleotide at the corresponding site of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to nucleotide sequence III of the target mRNA at the 5' end. Nucleotide sequence III is substantially reverse complementary or completely reverse complementary to nucleotide sequence II, or is a nucleotide sequence substantially reverse complementary or completely reverse complementary to the nucleotide sequence formed by nucleotide sequence II and nucleotide sequence IV.

In some embodiments, for the first siRNA, the sense strand of the miRNA comprises the nucleotide sequence set forth in SEQ ID NO: 5, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 6:

5'-CUUCAUUCAUCAGACAAZ ₃ -3' (SEQ ID NO: 5);

5'-Z ₄ UUGUCUGUAUGAAUGAAGAG-3' (SEQ ID NO: 6);

or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 7, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 8:

5'-CUCUUCAUUCAUCAGACAAZ ₃ -3' (SEQ ID NO: 7);

5'-Z ₄ UUGUCUGUAUGAAUGAAGAGAA-3' (SEQ ID NO: 8);

where Z ₄ represents the first nucleotide from the 5' end of the antisense strand; Z ₃ selected from A, U, G or C; and Z ₄ is a nucleotide complementary to Z ₃ .

In some embodiments, for miRNAs, the sense strand of the miRNA comprises the nucleotide sequence set forth in SEQ ID NO: 65, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 66:

5'-CUACAGUUUAGAAGAUUUZ ₇ -3' (SEQ ID NO: 65);

5'-Z ₈ AAAUCUUCUAAACUGUAGUA-3' (SEQ ID NO: 66),

or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 67, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 68:

5'-UACUACAGUUAGAAGAUUUZ ₇ -3' (SEQ ID NO: 67);

5'-Z ₈ AAAUCUUCUAAACUGUAGUAUG-3' (SEQ ID NO: 68),

where Z ₈ represents the first nucleotide from the 5' end of the antisense strand; Z ₇ selected from A, U, G or C; and Z ₈ is a nucleotide complementary to Z ₇ .

In some embodiments, for the third siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 125, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 126:

5'-GGAAGGUUACCGAGCAAUZ ₁₁ -3' (SEQ ID NO: 125);

5'-Z ₁₂ AUUGCUCGGUAACCUUCCCU-3' (SEQ ID NO: 126),

or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 127, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 128:

5'-AGGGAAGGUUACCGAGCAAUZ ₁₁ -3' (SEQ ID NO: 127);

5'-Z ₁₂ AUUGCUCGGUAACCUUCCCUGG-3' (SEQ ID NO: 128),

where Z ₁₂ represents the first nucleotide from the 5' end of the antisense strand; Z ₁₁ selected from A, U, G or C; and Z ₁₂ is a nucleotide complementary to Z ₁₁ .

In some embodiments, for the fourth miRNA, the sense strand of the miRNA comprises the nucleotide sequence set forth in SEQ ID NO: 185, and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 186:

5'-AGAACAGACAGCAGAUUZ ₁₅ -3' (SEQ ID NO: 185);

5'-Z ₁₆ AAUUCUGCUGUCUGUUCUCC-3' (SEQ ID NO: 186),

or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 187, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 188:

5'-GGAGAACAGACAGCAGAAUUZ ₁₅ -3' (SEQ ID NO: 187);

5'-Z ₁₆ AAUUCUGCUGUCUGUUCUCCUG-3' (SEQ ID NO: 188),

where Z ₁₆ represents the first nucleotide from the 5' end of the antisense strand; Z ₁₅ selected from A, U, G or C; and Z ₁₆ is a nucleotide complementary to Z ₁₅ .

In some embodiments, for the fifth siRNA, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 245 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 246:

5'-CCAAGAAGAACGCUGCAAZ ₁₉ -3' (SEQ ID NO: 245);

5'-Z ₂₀ UUGCAGCGUUCUUCUUGGCC-3' (SEQ ID NO: 246),

or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 247, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 248:

5'-GGCCAAGAAGAACGCUGCAAZ ₁₉ -3' (SEQ ID NO: 247);

5'-Z ₂₀ UUGCAGCGUUCUUCUUGGCCUG-3' (SEQ ID NO: 248),

where Z ₂₀ represents the first nucleotide from the 5' end of the antisense strand; Z ₁₉ selected from A, U, G or C; and Z ₂₀ is a nucleotide complementary to Z ₁₉ .

In some embodiments, for the sixth siRNA, the sense strand of the miRNA comprises the nucleotide sequence set forth in SEQ ID NO: 305 and the antisense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO: 306:

5'-CCAGUAAGCAAGCCAGAAZ ₂₃ -3' (SEQ ID NO: 305);

5'-Z ₂₄ UUCUGGCUUGCUUACUGGUA-3' (SEQ ID NO: 306),

or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 307, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 308:

5'-UACCAGUAAGCAAGCCAGAAZ ₂₃ -3' (SEQ ID NO: 307);

5'-Z ₂₄ UUCUGGCUUGCUUACUGGUAAC-3' (SEQ ID NO: 308),

where Z ₂₄ represents the first nucleotide from the 5' end of the antisense strand; Z ₂₃ selected from A, U, G or C; and Z ₂₄ is a nucleotide complementary to Z ₂₃ .

In some embodiments, the siRNA of the present invention is siC5a1, siC5a2, siC5b1, siC5b2, siC5c1, siC5c2, siC5d1, siC5d2, siC5e1, siC5e2, siC5f1, or siC5f2 listed in Tables 1a-1f.

As described above, each of the nucleotides in the siRNA according to the present invention is independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA of the present invention are unmodified nucleotides. In some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleotides. Such nucleotide modifications will not cause a significant reduction or loss of function of the siRNA of the present invention to inhibit C5 gene expression.

In some embodiments, the siRNA of the present invention comprises at least one modified nucleotide. As used herein, the term "modified nucleotide" as used herein refers to a nucleotide formed by replacing the 2'-hydroxy group of the ribose group of a nucleotide with other groups, a nucleotide analogue, or a nucleotide with a modified base. Such modified nucleotides will not cause a significant reduction or loss of miRNA function in inhibiting gene expression. For example, modified nucleotides may be selected as disclosed in Chemically Modified siRNA: tools and applications. Drug Discov Today, 2008.13(19-20): 842-55 per ed. J.K. Watts, G. F. Deleavey, and M. J. Damha.

In some embodiments, at least one nucleotide in the sense strand or antisense strand of a siRNA of the present invention is a modified nucleotide, and/or at least one phosphate is a modified phosphate group. In other words, at least a portion of the phosphate group and/or ribose group in the phosphate-ribose backbone of at least one single strand in the sense strand and the antisense strand is a group-modified phosphate group and/or a group-modified ribose group.

In some embodiments, all nucleotides in the sense strand and/or antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and antisense strand of the siRNA of the present invention is independently a fluorine-modified nucleotide or a nucleotide with a non-fluorine modification.

The inventors of the present invention have surprisingly found that the siRNA of the present invention has a high degree of balance between serum stability and gene silencing efficiency in animal experiments.

In some embodiments, the fluorine-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II; and in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of nucleotide sequence I are fluorine modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of nucleotide sequence ii are fluorine modified nucleotides.

In some embodiments, the fluorine-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II; there are no more than 5 fluorine modified nucleotides in the nucleotide sequence I, and in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 in the nucleotide sequence I are fluorine modified nucleotides; there are no more than 7 fluorine-modified nucleotides in nucleotide sequence II, and nucleotides at positions 2, 6, 14 and 16 in nucleotide sequence II are fluorine-modified nucleotides.

In some embodiments, in a 5' to 3' direction, the nucleotides at positions 7, 8, and 9 or 5, 7, 8, and 9 of nucleotide sequence I in the sense strand are fluorine modified nucleotides, and the nucleotides at the remaining positions in the sense chain are nucleotides with a modification other than fluorine; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 or 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand are fluorine modified nucleotides, and the nucleotides at the remaining positions in the antisense chain are nucleotides with a modification other than fluorine.

In the context of the present invention, the term “fluorine-modified nucleotide” refers to a nucleotide formed by replacing the 2'-hydroxy group of the ribose group of a nucleotide with fluorine, which has the structure represented by Formula (7). “Nucleotide with a non-fluorine modification” refers to a nucleotide formed by replacing the 2'-hydroxy group of the ribose group of a nucleotide with a non-fluorine group or nucleotide analogue. In some embodiments, each nucleotide with a non-fluorine modification is independently selected from a nucleotide formed by replacing the 2'-hydroxy group of the ribose group of the nucleotide with a non-fluorine group or a nucleotide analogue.

These nucleotides, formed by replacing the 2'-hydroxy group of the ribose group with a group other than fluorine, are well known to those skilled in the art, and these nucleotides can be selected from one of 2'-alkoxy-modified nucleotide, 2'-substituted alkoxy-modified nucleotide, 2'-alkyl-modified nucleotide, 2'-substituted alkyl-modified nucleotide, 2'-amino-modified nucleotide, 2'-substituted amino-modified nucleotide and 2'-deoxynucleotide.

In some embodiments, the 2'-alkoxy-modified nucleotide is a 2'-methoxy-modified nucleotide (2'-OMe) represented by Formula (8). In some embodiments, the 2'-substituted alkoxy-modified nucleotide is, for example, a 2'-O-methoxyethoxy-modified nucleotide (2'-MOE) represented by Formula (9). In some embodiments, the 2'-amino modified nucleotide (2'-NH ₂ ) is as represented by Formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is as represented by Formula (11):

A nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid and is structurally different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymidine deoxyribonucleotide. In some embodiments, the nucleotide analogue may be an isonucleotide, a bridged nucleic acid, or an acyclic nucleotide.

A bridging nucleic acid (BNA) is a nucleotide that is spatially restricted or inaccessible. BNA may contain a 5-membered ring, 6-membered ring, or bridged 7-membered ring structure having a sugar overhang in a “fixed” C3' endo conformation. The bridge is typically inserted at the 2' and 4' positions of the ribose, forming a 2',4'-BNA nucleotide. In some embodiments, the BNA may be an LNA, an ENA, and a cET BNA, wherein the LNA is as represented by Formula (12), the ENA is as represented by Formula (13), and the cET BNA is as represented by Formula (14):

An acyclic nucleotide is a nucleotide in which the ribose ring is open. In some embodiments, the acyclic nucleotide may be an unblocked nucleic acid (UNA) or a glycolic nucleic acid (GNA), where UNA is as represented by Formula (15) and GNA is as represented by Formula (16):

In Formula (15) and Formula (16), R is selected from H, OH or alkoxy (O-alkyl).

An isonucleotide is a compound formed by changing the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide may be a compound in which a base is moved from the -1' position to the -2' position or the -3' position on the ribose ring, as represented by Formulas (17) or (18):

In the compounds represented by Formula (17) and Formula (18) above, the Base is a base such as A, U, G, C or T; and R is selected from H, OH, F or a group other than fluorine described above.

In some embodiments, the nucleotide analog is selected from one of isonucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each nucleotide with a non-fluorine modification is a methoxy-modified nucleotide. In the context of the present invention, a methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxy group of a ribose group with a methoxy group.

In the context of the present invention, the terms "fluorine-modified nucleotide", "2'-fluoro-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced by fluorine" and "2'-fluororibosyl" have the same meaning and refer to the compound formed by replacing the 2'-hydroxy group of the ribose group of a nucleotide with fluorine, having the structure represented by Formula (7). The terms "methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "nucleotide in which the 2'-hydroxy group of the ribose group is replaced by methoxy" and "2'-methoxyribosyl" have the same meaning and refer to the compound formed by the substitution The 2'-hydroxy group of the ribose group of the nucleotide is a methoxy group having the structure represented by Formula (8).

In some embodiments, the siRNA of the present invention is an siRNA with the following modifications: in a 5' to 3' direction, the nucleotides at positions 7, 8, and 9 or 5, 7, 8, and 9 of nucleotide sequence I in the sense strand are are fluorine-modified nucleotides, and the nucleotides at other positions in the sense strand are methoxy-modified nucleotides; and nucleotides at positions 2, 6, 14 and 16 or 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand are fluorine-modified nucleotides, and nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides.

In some embodiments, the siRNA of the present invention is an siRNA with the following modifications: in a 5' to 3' direction, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at other positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are are methoxy-modified nucleotides;

or in a direction from the 5' end to the 3' end, nucleotides at positions 5, 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides;

or in a direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides.

In some embodiments, the siRNA of the present invention is any of siC5a1-M1, siC5a1-M2, siC5a1-M3, siC5a2-M1, siC5a2-M2, siC5a2-M3, siC5b1-M1, siC5b1-M2, siC5b1-M3, siC5b2- M1, siC5b2-M2, siC5b2-M3, siC5c1-M1, siC5c1-M2, siC5c1-M3, siC5c2-M1, siC5c2-M2, siC5c2-M3, siC5d1-M1, siC5d1-M2, siC5d1-M3, siC5d2-M1, siC5d2-M2, siC5d2-M3, siC5e1-M1, siC5e1-M2, siC5e1-M3, siC5e2-M1, siC5e2-M2, siC5e2-M3, siC5f1-M1, siC5f1-M2, siC5f1-M3, siC5f2-M1, siC5f2- M2 or siC5f2-M3 listed in Tables 1a-1f.

siRNAs with the above modifications not only can be produced at a lower cost, but also reduce the tendency of ribonucleases in the blood to degrade nucleic acid, thereby increasing the stability of the nucleic acid and making the nucleic acid more resistant to nuclease hydrolysis.

In some embodiments, at least a portion of the phosphate group in the phosphate-ribose backbone of at least one single strand in the sense strand and antisense strand of the siRNA of the present invention is a group-modified phosphate group. In some embodiments, the modified phosphate group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; and in some embodiments, the modified phosphate group is a thiophosphate group having the structure represented by Formula (1):

Formula 1).

This modification can stabilize the double-stranded structure of siRNA, thereby maintaining high specificity and high base-pairing affinity.

In some embodiments, in the siRNA of the present invention, the thiophosphate bond is present at at least one of the following positions: a position between the first nucleotide and the second nucleotide at either end of the sense strand or antisense strand; the position between the second and third nucleotides at either end of the sense strand or antisense strand; or any combination thereof. In some embodiments, a thiophosphate bond is present at all of the above positions except the 5' end of the sense strand. In some embodiments, a thiophosphate bond is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the thiophosphate bond is present at at least one of the following positions:

the position between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

the position between the second nucleotide and the third nucleotide at the 5' end of the sense strand;

the position between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

the position between the second nucleotide and the third nucleotide at the 3' end of the sense strand;

the position between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

the position between the second nucleotide and the third nucleotide at the 5' end of the antisense strand;

the position between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; And

the position between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

In some embodiments, the siRNA of the present invention is any of siC5a1-M1S, siC5a1-M2S, siC5a1-M3S, siC5a2-M1S, siC5a2-M2S, siC5a2-M3S, siC5b1-M1S, siC5b1-M2S, siC5b1-M3S, siC5b2- M1S, SIC5B2-M2S, SIC5B2-M3S, SIC5C1-M1S, SIC5C1-M2S, SIC5C1-M3S, SIC5C2-M1S, SIC5C2-M2S, SIC5C2-M3S, SIC5D1-M1S, SIC5D1-SIC5D1-M3S, SIC5D2-M1S, SIC siC5d2-M2S, siC5d2-M3S, siC5e1-M1S, siC5e1-M2S, siC5e1-M3S, siC5e2-M1S, siC5e2-M2S, siC5e2-M3S, siC5f1-M1S, siC5f1-M2S, siC5f1-M3S, siC5f2-M1S, siC5f 2- M2S or siC5f2-M3S listed in Tables 1a-1f.

In some embodiments, the 5' terminal nucleotide on the antisense strand of the siRNA is a nucleotide with a 5' phosphate group or a nucleotide modified with a 5' phosphate group analogue.

Common types of nucleotides with a 5'-phosphate group or nucleotides modified with a 5'-phosphate analogue are well known to those skilled in the art; for example, nucleotides with a 5'-phosphate group may have the following structure:

As another example, see The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 per ed. Anastasia Khvorova and Jonathan K. Watts, disclosed the following four nucleotides modified with an analogue of the 5'-phosphate group:

where R is selected from H, OH, methoxy or F; and The base is a base selected from A, U, C, G or T.

In some embodiments, the nucleotide with a 5'-phosphate group is the nucleotide with a 5'-phosphate modification represented by Formula (2); the 5'-phosphate analogue-modified nucleotide is a 5'-(E)-vinylphosphonate (E-VP) modified nucleotide represented by Formula (3) or a thiophosphate-modified nucleotide represented by Formula (5).

In some embodiments, the siRNA of the present invention is any of siC5a1-M1P1, siC5a1-M2P1, siC5a1-M3P1, siC5a2-M1P1, siC5a2-M2P1, siC5a2-M3P1, siC5b1-M1P1, siC5b1-M2P1, siC5b1-M3P1, siC5b2- M1P1, siC5b2-M2P1, siC5b2-M3P1, siC5c1-M1P1, siC5c1-M2P1, siC5c1-M3P1, siC5c2-M1P1, siC5c2-M2P1, siC5c2-M3P1, siC5d1-M1P1, siC5d1-M2P1, siC5d1-M3P 1, siC5d2-M1P1, siC5d2-M2P1, siC5d2-M3P1, siC5e1-M1P1, siC5e1-M2P1, siC5e1-M3P1, siC5e2-M1P1, siC5e2-M2P1, siC5e2-M3P1, siC5f1-M1P1, siC5f1-M2P1, siC5f1-M3P1, siC5 f2-M1P1, siC5f2- M2P1, siC5f2-M3P1, siC5a1-M1SP1, siC5a1-M2SP1, siC5a1-M3SP1, siC5a2-M1SP1, siC5a2-M2SP1, siC5a2-M3SP1, siC5b1-M1SP1, siC5b1-M2SP1, siC5b1-M3SP1, siC5b2-M1SP 1, siC5b2-M2SP1, siC5b2-M3SP1, siC5c1-M1SP1, siC5c1-M2SP1, siC5c1-M3SP1, siC5c2-M1SP1, siC5c2-M2SP1, siC5c2-M3SP1, siC5d1-M1SP1, siC5d1-M2SP1, siC5d1-M3SP1, siC5d2-M1SP1, siC5 d2-M2SP1, siC5d2- M3SP1, siC5e1-M1SP1, siC5e1-M2SP1, siC5e1-M3SP1, siC5e2-M1SP1, siC5e2-M2SP1, siC5e2-M3SP1, siC5f1-M1SP1, siC5f1-M2SP1, siC5f1-M3SP1, siC5f2-M1SP1, siC5f2-M2SP 1 or siC5f2-M3SP1, listed in Tables 1a-1f.

The inventors of the present invention have surprisingly found that the siRNA of the present invention has significantly increased stability in plasma and lysosomes and has a higher inhibitory activity against target mRNA.

The siRNA of the present invention can be produced using conventional methods for producing siRNA in the art (eg, solid phase synthesis methods and liquid phase synthesis methods). Commercial customization services are already available for solid-phase synthesis. Modified nucleotides can be introduced into siRNA according to the present invention using a nucleotide monomer having a suitable modification, where methods for producing a nucleotide monomer having a suitable modification and methods for introducing a modified nucleotide into siRNA are also well known to those skilled in the art. Modified nucleotide groups can be introduced into siRNA according to the present invention using a nucleotide monomer having a suitable modification, where methods for producing a nucleotide monomer having a suitable modification and methods for introducing the modified nucleotide into siRNA are also well known to those skilled in the art.

Pharmaceutical composition

According to the present invention, there is provided a pharmaceutical composition containing the siRNA described above as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be one commonly used in the field of siRNA administration, such as, but not limited to, one or more of magnetic nanoparticles (such as Fe ₃ O ₄ or Fe ₂ O ₃ -based nanoparticles), carbon nanotubes, mesoporous silicon, nanoparticles of calcium phosphate, polyethylenimine (PEI), polyamidoamine dendrimer (PAMAM), poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), D,L-lactic-glycolic copolymer acids (PLGA), poly(2-aminoethylethylene phosphate) (PPEEA), poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and their derivatives.

There are no special requirements for the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition, and it may be the usual content of each component. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier is 1:(1-500), and in some embodiments, the above weight ratio is 1:(1-50).

In some embodiments, the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventional in the art. For example, other pharmaceutically acceptable excipients may contain at least one of a pH buffer solution, a protecting agent, and an osmotic pressure regulator.

The pH buffer solution may be a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with pH = 7.5-8.5, and/or a phosphate buffer solution with pH = 5.5-8.5, preferably a phosphate buffer solution with pH = 5.5-8.5.

The protecting agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. The content of the protective agent may range from 0.01 wt.% to 30 wt.% of the total weight of the pharmaceutical composition.

The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator ensures the osmotic pressure of the pharmaceutical composition at a level of 200-700 milliosmol/kg (mOsm/kg). Depending on the target osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or lyophilized powder for injection, which is mixed with a liquid excipient to produce a liquid formulation for administration. The liquid formulation may be administered by, but not limited to, subcutaneous, intramuscular, or intravenous injection, and may be administered, but not limited to, to the lungs by nebulization or other organs (such as the liver) through the lungs by nebulization. In some embodiments, the pharmaceutical composition is administered by intravenous injection.

In some embodiments, the pharmaceutical composition may be in the form of a liposomal preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome preparation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid, and/or a PEGylated lipid. The organic amine, auxiliary lipid, and PEGylated lipid may be selected, respectively, from one or more of the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, auxiliary lipids, and PEGylated lipids described in CN103380113A (incorporated herein by reference in its entirety). volume).

In some embodiments, the organic amine may be the compound represented by Formula (201) described in reference CN103380113A, or a pharmaceutically acceptable salt thereof:

Formula (201),

Where:

X ₁₀₁ or X ₁₀₂ is independently selected from O, S, NA or CA, where A represents hydrogen or a C1-C20 hydrocarbon chain;

Y ₁₀₁ or Z ₁₀₁ is independently selected from C=O, C=S, S=O, CH-OH or SO ₂ ;

R ₁₀₁ , R ₁₀₂ , R ₁₀₃ , R ₁₀₄ , R ₁₀₅ , R ₁₀₆ or R ₁₀₇ are independently selected from hydrogen; cyclic or aliphatic, substituted or unsubstituted, branched or linear aliphatic group; cyclic or aliphatic, substituted or unsubstituted, branched or linear heteroaliphatic group; substituted or unsubstituted, branched or linear acyl group; substituted or unsubstituted, branched or linear aryl or substituted or unsubstituted, branched or linear heteroaryl;

x is an integer from 1 to 10;

n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1, where if m and p are both 0, then R ₁₀₂ is hydrogen, and

if at least one of n or m is 2, then R ₁₀₃ and nitrogen in Formula (201) form the structure represented by Formula (202) or (203):

where g, e or f independently represents an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom shown in Formula (201).

In some embodiments, R ₁₀₃ is a polyamine. In other embodiments, R ₁₀₃ is a ketal. In some embodiments, each of R ₁₀₁ and R ₁₀₂ in Formula (201) independently represents any substituted or unsubstituted, branched or straight alkyl or alkenyl, wherein said alkyl or alkenyl has from 3 to about 20 carbon atoms (e.g., from 8 to approximately 18 carbon atoms) and 0 to 4 double bonds (eg, 0 to 2 double bonds).

In some embodiments, if n and m are each independently equal to 1 to 3, R ₁₀₃ may be represented by any of the following Formulas (204)-(213):

where in Formula (204) - Formula (213), each of g, e and f independently represents an integer from 1 to 6; each "HCC" represents a hydrocarbon chain, and each * represents a potential point of attachment of R ₁₀₃ to a nitrogen atom in Formula (201), where each H at any marked * position can be substituted to effect attachment to a nitrogen atom in Formula (201) .

The compound shown in (201) can be prepared as described in reference CN103380113A.

In some embodiments, the organic amine may be an organic amine represented by Formula (214) and/or an organic amine represented by Formula (215):

Formula (214),

Formula (215).

The accessory lipid is cholesterol, a cholesterol analog and/or a cholesterol derivative.

The PEGylated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

In some embodiments, the molar ratio between the organic amine, the auxiliary lipid and the PEGylated lipid in the pharmaceutical composition is (19.7-80): (19.7-80): (0.3-50); for example, the molar ratio may be (50-70): (20-40): (3-20).

In some embodiments, the pharmaceutical compositions formed by the siRNA of the present invention and the above amine-containing transfection agent have an average diameter of from about 30 nm to about 200 nm, typically from about 40 nm to about 135 nm, and more often the average diameter of the liposome particles is from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 90 nm, or from about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.

In some embodiments, in the pharmaceutical composition formed by the siRNA of the present invention and the above amine-containing transfection agent, the weight ratio (w/w ratio) of the siRNA to the total amount of lipids (e.g., organic amine, auxiliary lipid, and/or PEGylated lipid) ranges from about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to about 1 :17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10. For example, the weight ratio of siRNA according to the present invention to total lipids is 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.

In some embodiments, each component in a pharmaceutical composition may be provided separately when marketed and the composition may be administered in the form of a liquid formulation. In some embodiments, the pharmaceutical composition formed by the siRNA of the present invention and the above pharmaceutically acceptable carrier can be prepared by various known methods other than replacing an existing siRNA with the siRNA of the present invention. In some embodiments, the pharmaceutical composition can be prepared in accordance with the following method.

Organic amines, auxiliary lipids and PEGylated lipids are suspended in alcohol at the molar ratio described above and mixed until homogeneous to obtain a lipid solution; and the alcohol is used in such an amount that the resulting lipid solution has a total mass concentration of 2 to 25 mg/ml, for example 8 to 18 mg/ml. The alcohol is a pharmaceutically acceptable alcohol, such as an alcohol that is in liquid form at about room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerin, PEG 200, PEG 300, PEG 400, and, for example, ethanol.

The siRNA of the present invention is dissolved in a buffered saline solution to obtain an aqueous siRNA solution. The buffered saline solution has a concentration of 0.05-0.5 M, for example 0.1-0.2 M. The pH of the buffered saline solution is adjusted to 4.0-5.5, for example 5.0-5.2. The buffered saline solution is used in such an amount that the siRNA is present at a concentration of less than 0.6 mg/ml, for example 0.2-0.4 mg/ml. The buffer salt may be one or more of the group consisting of soluble acetate and soluble citrate, such as sodium acetate and/or potassium acetate.

The lipid solution and the aqueous siRNA solution are mixed. The product obtained after mixing is incubated at a temperature of 40-60°C for at least 2 minutes (for example, 5-30 minutes) to obtain an incubated lipid-based drug. The volume ratio of lipid solution to aqueous siRNA solution is 1:(2-5).

The incubated liposome preparation is concentrated or diluted, purified to remove impurities and then sterilized to obtain a pharmaceutical composition according to the present invention having the following physicochemical parameters: pH 6.5-8, encapsulation percentage more than 80%, particle size 40-200 nm, polydispersity coefficient less than 0.30 and osmotic pressure 250-400 mOsm/kg; for example, the physicochemical parameters may be: pH 7.2-7.6, percent encapsulation greater than 90%, particle size 60-100 nm, polydispersity coefficient less than 0.20, and osmotic pressure 300-400 mOsm/kg.

The concentration or dilution step may be carried out before, after or simultaneously with the impurity removal step. The impurity removal method may be any of various existing methods, such as ultrafiltration using a 100 kDa hollow fiber column and phosphate buffer saline (PBS) at pH = 7.4 as a replacement ultrafiltration solution, and a tangential flow system. The sterilization method may be any of various existing methods, such as sterilizing filtration on a filter with a pore size of 0.22 microns.

siRNA conjugate

The present invention relates to an siRNA conjugate containing the above siRNA and a conjugating group linked by conjugation to the specified siRNA.

The conjugating group typically contains at least one pharmaceutically acceptable targeting group and an optional linker. In addition, the siRNA, linker and targeting group are connected in series. In some embodiments, 1-6 targeting groups are present. In some embodiments, 2-4 targeting groups are present. The siRNA molecule may be non-covalently or covalently conjugated to a conjugating group, for example, the siRNA molecule may be covalently conjugated to a conjugating group. The conjugation site of the siRNA and the conjugating group may be located at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugating group is located at the 3' end of the sense strand of the siRNA.

In some embodiments, the conjugating group is linked to a phosphate group, a 2'-hydroxy group, or a nucleotide base. In some embodiments, the conjugating group may be linked to a 3'-hydroxy when the nucleotides are linked through a 2'-5' phosphodiester bond. If a conjugating group is linked to the end of the siRNA, the conjugating group is typically linked to the phosphate group of the nucleotide; if the conjugating group is linked to an internal sequence of the siRNA, the conjugating group is typically linked to a ribose ring or base. Specific binding options are given in the source siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides identify robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5):1181-7, per ed. Muthiah Manoharan et al.

In some embodiments, the siRNA and the conjugating group may be linked through an acid-labile or reducible chemical bond, and these chemical bonds may be disrupted in the acidic environment of the cell's endosomes, rendering the siRNA free. For non-degradable conjugation options, the conjugating group can be linked to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutically acceptable targeting moiety may be selected from one or more of the ligands formed by the following targeting molecules or derivatives thereof: lipophilic molecules such as cholesterol, bile acids, vitamins (such as vitamin E), lipid molecules of various chain lengths ; polymers such as polyethylene glycol; polypeptides such as penetrating peptide; aptamers; antibodies; quantum dots; saccharides such as lactose, polylactose, mannose, galactose and N-acetylgalactosamine (GalNAc); folate; and receptor ligands expressed in liver parenchyma cells, such as asialoglycoprotein, asialosugar moiety, lipoproteins (such as high-density lipoprotein, low-density lipoprotein), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin and the like.

In some embodiments, each ligand is independently a ligand capable of binding to a cell surface receptor. In some embodiments, the at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, the at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, the at least one ligand is a ligand capable of binding to a human cell surface receptor. In some embodiments, the at least one ligand is a ligand capable of binding to an asialoglycoprotein receptor (ASGPR) on the surface of a hepatocyte. The types of these ligands are well known to those skilled in the art, and they typically have the function of binding to certain receptors on the surface of a target cell, mediating the delivery of siRNA bound to the ligand into the target cell.

In some embodiments, the pharmaceutically acceptable targeting moiety can be any ligand that binds to asialoglycoprotein receptors (ASGPRs) on the surface of mammalian hepatocytes. In one embodiment, each ligand is independently an asialoglycoprotein, such as asialorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a saccharide or a saccharide derivative.

In some embodiments, the at least one ligand is a saccharide. In some embodiments, each ligand is a saccharide. In some embodiments, the at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a saccharide derivative. In some embodiments, the at least one ligand may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, the at least one ligand is a modified saccharide. In some embodiments, each ligand is a modified saccharide. In some embodiments, each ligand is independently selected from the group consisting of polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each ligand or at least one ligand is selected from the group consisting of the following saccharides: glucose and a derivative thereof, mannose and a derivative thereof, galactose and a derivative thereof, xylose and a derivative thereof, ribose and a derivative thereof, fucose and a derivative thereof , lactose and a derivative thereof, maltose and a derivative thereof, arabinose and a derivative thereof, fructose and a derivative thereof, and sialic acid.

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

In some embodiments, the pharmaceutically acceptable targeting moiety in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecules may be monovalent, divalent, trivalent, or tetravalent. It should be understood that the terms “monivalent,” “divalent,” “trivalent,” and “tetravalent” as used herein respectively mean that the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1 , 1:2, 1:3 or 1:4, where the siRNA conjugate is formed from a siRNA molecule and a conjugating group containing galactose or N-acetylgalactosamine as a targeting group. In some embodiments, the pharmaceutically acceptable targeting moiety is N-acetylgalactosamine. In some embodiments, when the siRNA of the present invention is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA of the present invention is conjugated to a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

The targeting group can be linked to the siRNA molecule through a suitable linker, and said suitable linker can be selected by those skilled in the art according to the particular type of targeting group. The types of these linkers and targeting groups, as well as methods for binding to siRNAs, can be found in WO2015006740A2, which is incorporated herein by reference in its entirety.

In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be the structure represented by Formula (301):

Formula (301),

Where

k is an integer from 1 to 3; And

L ^A is an amide bond-containing chain moiety having the structure represented by Formula (302), with each L ^A suitably linked to a targeting group and an L ^C moiety via an ether bond at its two ends:

Formula (302);

L ^B is a chain moiety containing N-acylpyrrolidine having the structure represented by Formula (303), wherein the chain moiety contains a carbonyl at one end thereof and is linked to the L ^C moiety via an amide bond, and contains an hydroxy group at the other end thereof and is linked to siRNA via phosphoester linkage:

Formula (303);

L ^C is a di- to tetravalent linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane, wherein L ^C is linked to each of the L ^A moieties via an ether bond via an oxygen atom and linked to the L ^B moiety via an amide bond via a nitrogen atom.

In some embodiments, when n=3 and L ^C is a tetravalent trihydroxymethylaminomethane-based linking group, the siRNA conjugate formed by linking an N-acetylgalactosamine molecule to a siRNA molecule using -(L ^A ) ₃ -trihydroxymethylaminomethane-L ^B - as linker, has the structure represented by Formula (304):

Formula (304),

where the double helix structure is siRNA.

Likewise, the conjugation site of the siRNA and the conjugating group may be located at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA.

In some embodiments, the 3' end of the sense strand of an siRNA of the present invention is covalently conjugated to three molecules of N-acetylgalactosamine (GalNAc) via a linker -(L ^A ) ₃ -trihydroxymethylaminomethane-L ^B - to produce an siRNA conjugate in which the molar ratio of the molecule The siRNA to GaINAc molecule is 1:3, which may also be referred to herein as (GaINAc) ₃ -siRNA), and the siRNA conjugate has the structure represented by Formula (305):

Formula (305),

wherein the double helix structure is siRNA; and the linker is connected to the 3' end of the sense strand of the siRNA.

In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be the structure represented by Formula (306):

, Formula (306),

Where

l represents an integer from 0 to 3;

* denotes a site linked to the targeting group via an ether bond on the linker; And

# denotes the site linked to the siRNA via a phosphoester bond on the linker.

In some embodiments, when l=2, the siRNA conjugate has the structure represented by Formula (307):

Formula (307),

wherein the double helix structure is siRNA; and the linker is connected to the 3' end of the sense strand of the siRNA.

The above conjugates can be synthesized in accordance with methods described in detail in the prior art. For example, WO2015006740A2 describes in detail a method for preparing various conjugates. The siRNA conjugate of the present invention can be prepared by methods well known to those skilled in the art. As another example, WO2014025805A1 describes a method for preparing a conjugate having the structure represented by Formula (305). Rajeev et al. describe a method for preparing a conjugate having the structure represented by Formula (307) in ChemBioChem 2015, 16, 903-908.

In some embodiments, the siRNA conjugate has the structure represented by Formula (308):

Formula (308),

Where:

n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4;

each of m1, m2 and m3 independently represents an integer from 2 to 10;

each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ or R ₁₅ independently represents H or is selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl and C ₁ -C ₁₀ alkoxy ; And

R ₃ is a group having the structure represented by Formula A59:

(A59),

where E ₁ represents OH, SH or BH ₂ and Nu represents siRNA according to the present invention;

R ₂ is a linear alkylene of 1 to 20 carbon atoms in length, where one or more carbon atoms are optionally substituted by any one or more of the group consisting of: C(O), NH, O, S, CH=N, S(O ) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene, and where R ₂ is optionally substituted by any one or more of the group , consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl-NH ₂ , -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -NH(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkylphenyl), -NH(C ₁ -C ₁₀ alkylphenyl), cyano, nitro, -CO ₂ H, -C(O)O( C ₁ -C ₁₀ alkyl), -CON(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ alkyl)C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)C( O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ - C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH(phenyl), -NHSO ₂ (C ₁ -C ₁₀ alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl); And

each L ₁ is independently a linear alkylene of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally substituted by any one or more of the group consisting of: C(O), NH, O, S, CH=N, S (O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein L ₁ is optionally substituted by any one or more of the group consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl-NH ₂ , -N(C ₁ -C ₁₀ alkyl)(C ₁ - C ₁₀ alkyl), -NH(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkylphenyl), -NH(C ₁ -C ₁₀ alkylphenyl), cyano, nitro, -CO ₂ H, -C(O)O(C ₁ -C ₁₀ alkyl), -CON(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ alkyl)C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)C(O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ - C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH (phenyl), -NHSO ₂ (C ₁ -C ₁₀ alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C ₁ - C ₁₀ haloalkyl).

In some embodiments, L ₁ may be selected from the group consisting of groups (A1)-(A26) and any combination thereof, wherein the structures and definitions (A1)-(A26) are as follows:

where each j1 independently represents an integer from 1 to 20; and each j2 is independently an integer from 1 to 20;

each R' independently represents C ₁ -C ₁₀ alkyl; And

each Ra is independently selected from the group consisting of groups (A27)-(A45) and any combinations of their compounds:

each Rb independently represents C ₁ -C ₁₀ alkyl; And denotes the site of covalent addition of a group.

Those skilled in the art will appreciate that while L ₁ is defined as a linear alkylene for convenience, it may not be a linear group or may be called something else, such as an amine or an alkenyl derived from the above substitution and/ or substitution. For the purposes of the present invention, the length L ₁ represents the number of atoms in the chain connecting two points of attachment. For this purpose, a ring obtained by replacing a carbon atom of a linear alkylene such as heterocyclylene or heteroarylene is counted as one atom.

M ₁ is a targeting group, the definitions and variations of which are similar to those described above. In some embodiments, each M ₁ is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian hepatocytes.

When M ₁ is a ligand having affinity for an asialoglycoprotein receptor on the surface of mammalian hepatocytes, in some embodiments n1 may be an integer from 1 to 3 and n3 may be an integer from 0 to 4 such that the number of targeting groups M ₁ in the siRNA conjugate could be at least 2. In some embodiments, n1+n3≥2, such that the number of M ₁ targeting moieties in the conjugate can be at least 3, thereby allowing for easier binding of the M ₁ targeting moiety to the asialoglycoprotein receptor on surface of hepatocytes, which may facilitate endocytosis of the siRNA conjugate into cells. Experiments have shown that when the number of M ₁ targeting groups exceeds 3, the ease of binding of the M ₁ targeting group to the asialoglycoprotein receptor on the surface of hepatocytes does not increase significantly. Thus, taking into account various aspects such as ease of synthesis, process design/cost, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+ n3=2-3.

In some embodiments, when m1, m2, or m3 are each independently selected from an integer of 2 to 10, the relative spatial arrangements of the plurality of M ₁ targeting groups may be suitable for binding the M ₁ targeting groups to the asialoglycoprotein receptor on the surface of hepatocytes. In order for the siRNA conjugate of the present invention to have a simpler structure, easier synthesis, and/or reduced cost, in some embodiments, m1, m2, or m3 is independently an integer from 2 to 5, and in some embodiments, m1=m2 =m3.

Those skilled in the art will appreciate that when each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ or R ₁₅ is independently selected from H, C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ haloalkyl and C ₁ -C ₁₀ alkoxy, they do not change the properties of the siRNA conjugate according to the present invention and are all capable of achieving the purpose of the present invention. In some embodiments, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ or R ₁₅ are each independently selected from H, methyl or ethyl. In some embodiments, _R10 , _R11 , _R12 , _R13 , _R14 , and _R15 are all H.

R ₃ represents a group having the structure represented by Formula A59, where E ₁ represents OH, SH or BH ₂ , and when the starting materials are available, in some embodiments, E ₁ represents OH or SH.

R ₂ is chosen to provide a bond between the group represented by Formula A59 and the N atom on the nitrogen backbone. In the context of the present invention, “nitrogen-containing backbone” refers to a chain structure in which the carbon atoms attached to R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ and the N atoms are connected to each other. Therefore, R ₂ can be any linking group capable of attaching the group represented by Formula A59 to the N atom on the nitrogen backbone by suitable means. In some embodiments, when the siRNA conjugate represented by Formula (308) according to the present invention is prepared by a solid-phase synthesis process, the R ₂ group must have both a binding site to the N atom on the nitrogen backbone and a binding site to the N atom P to _R3 . In some embodiments, in R ₂ the binding site to the N atom on the nitrogen backbone forms an amide bond to the N atom, and the binding site to the P atom in R ₃ forms a phosphoester bond to the P atom. In some embodiments, R ₂ may be B5, B6 , B5' or B6':

Where denotes the site of covalent attachment of a group; And

the range of q ₂ values may be an integer from 1 to 10; and in some embodiments, q ₂ is an integer from 1 to 5.

L ₁ is used to bind the targeting group M ₁ to the N atom on the nitrogen backbone, which provides the siRNA conjugate represented by Formula (308) with liver targeting function. In some embodiments, L ₁ is selected from combinations of compounds from one or more of the groups represented by Formulas A1-A26. In some embodiments, L ₁ is selected from combinations of compounds of one or more of A1, A4, A5, A6, A8, A10, A11, and A13. In some embodiments, L ₁ is selected from combinations of compounds of at least two of A1, A4, A8, A10, and A11. In some embodiments, L ₁ is selected from combinations of compounds of at least two of A1, A8 and A10.

In some embodiments, the length of L ₁ may be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms. In some embodiments, the length L ₁ is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms.

In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is is an integer from 3 to 5. R' is C ₁ -C ₄ alkyl, and in some embodiments, R' is one of methyl, ethyl and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is C ₁ -C ₅ alkyl, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra and Rb of Formulas A1-A26 are appropriately selected to provide binding between the targeting group M ₁ and the N atom on the nitrogen backbone, as well as to create a relative spatial arrangement of the targeting groups M ₁ , more suitable for binding the M ₁ targeting group to the asialoglycoprotein receptor on the surface of hepatocytes.

In some embodiments, the siRNA conjugate has the structure represented by Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412) , (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422):

Formula (403)

Formula (404)

Formula (405)

Formula (406)

Formula (407)

Formula (408)

Formula (409)

Formula (410)

Formula (411)

Formula (412)

Formula (413)

Formula (414)

Formula (415)

Formula (416)

Formula (417)

Formula (418)

Formula (419)

Formula (420)

Formula (421)

Formula (422).

In some embodiments, the P atom in Formula A59 can be linked to any possible position in the siRNA sequence, for example, the P atom in Formula A59 can be linked to any nucleotide in the sense strand or antisense strand of the siRNA. In some embodiments, the P atom in Formula A59 is linked to any nucleotide in the sense strand of the siRNA. In some embodiments, the P atom in Formula A59 is associated with the end of the sense strand or antisense strand of the siRNA. In some embodiments, the P atom in Formula A59 is associated with the end of the siRNA sense strand. The end refers to the first 4 nucleotides counted from one end of the sense strand or antisense strand. In some embodiments, the P atom in Formula A59 is associated with the end of the sense strand or antisense strand of the siRNA. In some embodiments, the P atom in Formula A59 is associated with the 3' end of the siRNA sense strand. In case the P atom in Formula A59 is associated with the above position in the siRNA sense strand, after entering the cells, the siRNA conjugate represented by Formula (308) can release a separate antisense strand of the siRNA during unwinding, thereby blocking the translation of C5 mRNA into protein and inhibiting the expression of the complement protein gene C5.

In some embodiments, the P atom in Formula A59 may be associated with any possible nucleotide position in the siRNA, such as the 5', 2', or 3' position, or the base of the nucleotide. In some embodiments, the P atom in Formula A59 may be linked to a 2', 3', or 5' nucleotide position in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in Formula A59 is bonded to an oxygen atom formed by deprotonation of the 3'-hydroxy group of the nucleotide at the 3' end of the sense strand of the siRNA (at which time, the P atom in Formula A59 may also be considered the P atom in the phosphate group, contained in siRNA), or the P atom in Formula A59 is bonded to the nucleotide by replacing a hydrogen atom in the 2'-hydroxy group of the nucleotide of the sense strand of the siRNA, or the P atom in Formula A59 is bonded to the nucleotide by replacing the hydrogen in the 5'-hydroxy group of the nucleotide with a 5'- end of the sense strand of the miRNA.

The inventors of the present invention have surprisingly found that the siRNA conjugate of the present invention has significantly improved plasma stability and low off-target activity, and also exhibits higher C5 mRNA silencing activity. In some embodiments, the miRNA of the present invention may be one of the miRNAs presented in Tables 1a-1f. SiRNA conjugates containing these siRNAs exhibit higher silencing activity against C5 mRNA.

Table 1a. Sequence of the first miRNA according to the present invention

miRNA no. SEQ ID NO: Sequence recording direction 5’- 3’ siC5a1 9 CUUCAUUCAUACAGACAAA 10 UUUGUCUGUAUGAAUGAAGAG siC5a2 eleven CUCUUCAUUCAUACAGACAAA 12 UUUGUCUGUAUGAAUGAAGAGAA siC5a1-M1 13 CmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 14 UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm siC5a1-M2 15 CmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 16 UmUfUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGm siC5a1-M3 17 CmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 18 UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm siC5a2-M1 19 CmUmCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 20 UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm siC5a2-M2 21 CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 22 UmUfUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmAmAm siC5a2-M3 23 CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 24 UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm siC5a1-M1S 25 CmsUmsUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 26 UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm siC5a1-M2S 27 CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 28 UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmsAmsGm siC5a1-M3S 29 CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm thirty UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm siC5a2-M1S 31 CmsUmsCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 32 UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm siC5a2-M2S 33 CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 34 UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm siC5a2-M3S 35 CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 36 UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm siC5a1-M1P1 37 CmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 38 P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm siC5a1-M2P1 39 CmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 40 P1UmUfUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGm siC5a1-M3P1 41 CmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 42 P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm siC5a2-M1P1 43 CmUmCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 44 P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm siC5a2-M2P1 45 CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 46 P1UmUfUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmAmAm siC5a2-M3P1 47 CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 48 P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm siC5a1-M1SP1 49 CmsUmsUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 50 P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm siC5a1-M2SP1 51 CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 52 P1UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmsAmsGm siC5a1-M3SP1 53 CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 54 P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm siC5a2-M1SP1 55 CmsUmsCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 56 P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm siC5a2-M2SP1 57 CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 58 P1UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm siC5a2-M3SP1 59 CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm 60 P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm

Table 1b. Sequence of the second siRNA according to the present invention

miRNA no. SEQ ID NO: Sequence recording direction 5’- 3’ siC5b1 69 CUACAGUUUAGAAGAUUUA 70 UAAAUCUUCUAAACUGUAGUA siC5b2 71 UACUACAGUUUAGAAGAUUUA 72 UAAAUCUUCUAAACUGUAGUAUG siC5b1-M1 73 CmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 74 UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm siC5b1-M2 75 CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 76 UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAm siC5b1-M3 77 CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 78 UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm siC5b2-M1 79 UmAmCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 80 UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm siC5b2-M2 81 UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 82 UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmUmGm siC5b2-M3 83 UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 84 UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm siC5b1-M1S 85 CmsUmsAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 86 UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm siC5b1-M2S 87 CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 88 UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmsUmsAm siC5b1-M3S 89 CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 90 UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm siC5b2-M1S 91 UmsAmsCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 92 UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm siC5b2-M2S 93 UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 94 UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm siC5b2-M3S 95 UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 96 UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm siC5b1-M1P1 97 CmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 98 P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm siC5b1-M2P1 99 CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 100 P1UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAm siC5b1-M3P1 101 CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 102 P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm siC5b2-M1P1 103 UmAmCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 104 P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm siC5b2-M2P1 105 UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 106 P1UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmUmGm siC5b2-M3P1 107 UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 108 P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm siC5b1-M1SP1 109 CmsUmsAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 110 P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm siC5b1-M2SP1 111 CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 112 P1UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmsUmsAm siC5b1-M3SP1 113 CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 114 P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm siC5b2-M1SP1 115 UmsAmsCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 116 P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm siC5b2-M2SP1 117 UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 118 P1UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm siC5b2-M3SP1 119 UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm 120 P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm

Table 1c. Sequence of the third siRNA according to the present invention

miRNA no. SEQ ID NO: Sequence recording direction 5’- 3’ siC5c1 129 GGAAGGUUACCGAGCAAUA 130 UAUUGCUCGGUAACCUUCCCU siC5c2 131 AGGGAAGGUUACCGAGCAAUA 132 UAUUGCUCGGUAACCUUCCCUGG siC5c1-M1 133 GmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 134 UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm siC5c1-M2 135 GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 136 UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUm siC5c1-M3 137 GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 138 UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm siC5c2-M1 139 AmGmGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 140 UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm siC5c2-M2 141 AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 142 UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmGmGm siC5c2-M3 143 AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 144 UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm siC5c1-M1S 145 GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 146 UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm siC5c1-M2S 147 GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 148 UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmsCmsUm siC5c1-M3S 149 GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 150 UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm siC5c2-M1S 151 AmsGmsGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 152 UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm siC5c2-M2S 153 AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 154 UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm siC5c2-M3S 155 AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 156 UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm siC5c1-M1P1 157 GmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 158 P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm siC5c1-M2P1 159 GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 160 P1UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUm siC5c1-M3P1 161 GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 162 P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm siC5c2-M1P1 163 AmGmGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 164 P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm siC5c2-M2P1 165 AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 166 P1UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmGmGm siC5c2-M3P1 167 AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 168 P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm siC5c1-M1SP1 169 GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 170 P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm siC5c1-M2SP1 171 GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 172 P1UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmsCmsUm siC5c1-M3SP1 173 GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 174 P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm siC5c2-M1SP1 175 AmsGmsGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 176 P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm siC5c2-M2SP1 177 AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 178 P1UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm siC5c2-M3SP1 179 AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm 180 P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm

Table 1d. Sequence of the fourth miRNA according to the present invention

miRNA no. SEQ ID NO: Sequence recording direction 5’- 3’ siC5d1 189 AGAACAGACAGCAGAAUUA 190 UAAUUCUGCUGUCUGUUCUCC siC5d2 191 GGAGAACAGACAGCAGAAUUA 192 UAAUUCUGCUGUCUGUUCUCCUG siC5d1-M1 193 AmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 194 UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm siC5d1-M2 195 AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 196 UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCm siC5d1-M3 197 AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 198 UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm siC5d2-M1 199 GmGmAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 200 UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm siC5d2-M2 201 GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 202 UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmUmGm siC5d2-M3 203 GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 204 UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm siC5d1-M1S 205 AmsGmsAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 206 UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm siC5d1-M2S 207 AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 208 UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmsCmsCm siC5d1-M3S 209 AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 210 UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm siC5d2-M1S 211 GmsGmsAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 212 UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm siC5d2-M2S 213 GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 214 UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm siC5d2-M3S 215 GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 216 UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm siC5d1-M1P1 217 AmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 218 P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm siC5d1-M2P1 219 AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 220 P1UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCm siC5d1-M3P1 221 AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 222 P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm siC5d2-M1P1 223 GmGmAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 224 P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm siC5d2-M2P1 225 GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 226 P1UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmUmGm siC5d2-M3P1 227 GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 228 P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm siC5d1-M1SP1 229 AmsGmsAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 230 P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm siC5d1-M2SP1 231 AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 232 P1UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmsCmsCm siC5d1-M3SP1 233 AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 234 P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm siC5d2-M1SP1 235 GmsGmsAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 236 P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm siC5d2-M2SP1 237 GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 238 P1UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm siC5d2-M3SP1 239 GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm 240 P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm

Table 1e. Sequence of the fifth miRNA according to the present invention

miRNA no. SEQ ID NO: Sequence recording direction 5’- 3’ siC5e1 249 CCAAGAGAACGCUGCAAA 250 UUUGCAGCGUUCUUCUUGGCC siC5e2 251 GGCCAAGAAGAACGCUGCAAA 252 UUUGCAGCGUUCUUCUUGGCCUG siC5e1-M1 253 CmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 254 UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm siC5e1-M2 255 CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 256 UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCm siC5e1-M3 257 CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 258 UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm siC5e2-M1 259 GmGmCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 260 UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm siC5e2-M2 261 GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 262 UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmUmGm siC5e2-M3 263 GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 264 UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm siC5e1-M1S 265 CmsCmsAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 266 UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm siC5e1-M2S 267 CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 268 UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmsCmsCm siC5e1-M3S 269 CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 270 UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm siC5e2-M1S 271 GmsGmsCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 272 UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm siC5e2-M2S 273 GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 274 UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm siC5e2-M3S 275 GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 276 UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm siC5e1-M1P1 277 CmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 278 P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm siC5e1-M2P1 279 CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 280 P1UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCm siC5e1-M3P1 281 CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 282 P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm siC5e2-M1P1 283 GmGmCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 284 P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm siC5e2-M2P1 285 GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 286 P1UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmUmGm siC5e2-M3P1 287 GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 288 P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm siC5e1-M1SP1 289 CmsCmsAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 290 P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm siC5e1-M2SP1 291 CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 292 P1UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmsCmsCm siC5e1-M3SP1 293 CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 294 P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm siC5e2-M1SP1 295 GmsGmsCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 296 P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm siC5e2-M2SP1 297 GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 298 P1UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm siC5e2-M3SP1 299 GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm 300 P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm

Table 1f. Sequence of the sixth miRNA according to the present invention

miRNA no. SEQ ID NO: Sequence recording direction 5’- 3’ siC5f1 309 CCAGUAAGCAAGCCAGAAA 310 UUUCUGGCUUGCUUACUGGUA siC5f2 311 UACCAGUAAGCAAGCCAGAAA 312 UUUCUGGCUUGCUUACUGGUAAC siC5f1-M1 313 CmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 314 UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm siC5f1-M2 315 CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 316 UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAm siC5f1-M3 317 CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 318 UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm siC5f2-M1 319 UmAmCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 320 UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm siC5f2-M2 321 UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 322 UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmAmCm siC5f2-M3 323 UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 324 UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm siC5f1-M1S 325 CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 326 UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm siC5f1-M2S 327 CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 328 UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmsUmsAm siC5f1-M3S 329 CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 330 UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm siC5f2-M1S 331 UmsAmsCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 332 UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm siC5f2M2S 333 UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 334 UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm siC5f2-M3S 335 UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 336 UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm siC5f1-M1P1 337 CmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 338 P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm siC5f1-M2P1 339 CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 340 P1UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAm siC5f1-M3P1 341 CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 342 P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm siC5f2-M1P1 343 UmAmCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 344 P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm siC5f2-M2P1 345 UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 346 P1UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmAmCm siC5f2-M3P1 347 UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 348 P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm siC5f1-M1SP1 349 CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 350 P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm siC5f1-M2SP1 351 CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 352 P1UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmsUmsAm siC5f1-M3SP1 353 CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 354 P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm siC5f2-M1SP1 355 UmsAmsCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 356 P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm siC5f2-M2SP1 357 UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 358 P1UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm siC5f2-M3SP1 359 UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm 360 P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm

In the siRNA or siRNA conjugate of the present invention, each pair of adjacent nucleotides is linked by a phosphodiester bond or a thiophosphate bond. The non-bridging oxygen atom or sulfur atom in a phosphodiester bond or thiophosphate bond is negatively charged and may be present in the form of a hydroxy group or a sulfhydryl group. In addition, the hydrogen ion in the hydroxy group or sulfhydryl group may be partially or completely replaced by a cation. The cation may be any cation such as a metal cation, an ammonium ion NH4 ⁺ or an organic ammonium cation. To increase solubility, in some embodiments, the cation is selected from one or more of an alkali metal ion, a tertiary amine ammonium cation, and a quaternary ammonium cation. The alkali metal ion may be K ⁺ and/or Na ⁺ , and the tertiary amine cation may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N,N-diisopropylethylamine. Thus, the siRNA or siRNA conjugate of the present invention may be at least partially present in salt form. In one embodiment, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphate bond is at least partially bound to a sodium ion, and thus the siRNA or siRNA conjugate of the present invention is present or partially present in the form of a sodium salt.

It is well known to those skilled in the art that a modified nucleotide group can be introduced into the siRNA according to the present invention using a nucleoside monomer having a suitable modification. Methods for preparing a nucleoside monomer having a suitable modification and methods for introducing a modified nucleotide group into siRNA are also well known to those skilled in the art. All modified nucleoside monomers may be commercially available or prepared by known methods.

Preparation of siRNA conjugate represented by Formula (308)

The siRNA conjugate represented by Formula (308) can be prepared by any suitable synthetic route.

In some embodiments, the siRNA conjugate represented by Formula (308) can be prepared using the following method. The method includes: sequential binding of nucleoside monomers in the direction from 3' to 5' in accordance with the types and sequences of nucleotides in the sense chain and antisense chain, respectively, under the conditions of solid-phase amidophosphite synthesis, where the stage of binding each nucleoside monomer includes a four-stage deprotection reaction, combination , capping and oxidation or sulfurization; isolation of the sense strand and antisense strand of siRNA; and annealing; wherein the siRNA is the siRNA of the present invention mentioned above.

In addition, the method further includes: bringing the compound represented by Formula (321) into contact with a nucleoside monomer or nucleotide sequence bound to a solid phase support under coupling reaction conditions and in the presence of a coupling agent, causing binding of the compound represented by Formula (321) , with a nucleotide sequence through a coupling reaction. Hereinafter, the compound represented by Formula (321) is also referred to as a conjugating molecule.

Formula (321),

Where:

R ₄ represents a group capable of binding to the siRNA represented by Nu in the compound represented by Formula (308). In some embodiments, R ₄ is a group capable of binding to the siRNA represented by Nu through a covalent bond. In some embodiments, R ₄ is a group capable of being conjugated to any siRNA functional group represented by Nu via a phosphodiester bond during the reaction;

each S ₁ independently represents M ₁ , which is a group formed by replacing all active hydroxy groups with YCOO-, where each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl , isopropyl, phenyl, halophenyl and alkylphenyl. In some embodiments, Y is methyl.

The definitions and options n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L ₁ and M ₁ , respectively, are as described above.

R ₄ is selected to bind to the N atom on the nitrogen backbone and provide a suitable reaction site for the synthesis of the siRNA conjugate represented by Formula (308). In some embodiments, R ₄ contains an R ₂ linking group or a protected R ₂ linking group and can form the functional group represented by Formula (A59) with siRNA through reaction.

In some embodiments, R4 contains a first functional group that can react with a group on the siRNA or nucleoside monomer represented by Nu to form a phosphite ester, and a second functional group that can form a covalent bond with a hydroxy group or an amino group or contains a solid phase support linked by covalent bond. In some embodiments, the first functional group is an amidophosphite, a hydroxy group, or a protected hydroxy group. In some embodiments, the second functional group is an amidophosphite, carboxyl, or carboxylate. In some embodiments, the second functional group is a solid phase support linked to the rest of the molecule through a covalent bond formed by a hydroxy group or an amino group. In some embodiments, the solid phase support is linked through a phosphoester bond, a carboxyester bond, or an amide bond. In some embodiments, the solid phase support is a resin.

In some embodiments, the first functional group contains a hydroxy group, -OR _k or a group represented by Formula (C3); and the second functional group contains a group represented by Formula (C1), (C2), (C3), (C1') or (C3'):

where q ₁ is an integer from 1 to 4, X is O or NH, M ⁺ is a cation, R _k is a hydroxy protecting group, TPP is a solid phase support, and denotes the site of covalent addition of a group.

In some embodiments, the first functional group contains an amidophosphite group represented by Formula (C3). The amidophosphite group can form a phosphite ester with a hydroxy group at any position on the nucleotide, such as a 2'- or 3'-hydroxy group, through a coupling reaction, and the phosphite ester can form a phosphodiester bond or a thiophosphate bond represented by Formula (A59) through oxidation or sulfurization to conjugate the conjugating molecule to the siRNA. In this case, even if the second functional group does not exist, the compound represented by Formula (321) can still be conjugated to a nucleotide without affecting the production of the siRNA conjugate represented by Formula (308). In such circumstances, after obtaining the sense strand or antisense strand of siRNA by a method such as solid-phase amidophosphite synthesis, the compound represented by Formula (321) is reacted with a hydroxy group at the terminal nucleotide of the nucleotide sequence, and a phosphodiester bond or thiophosphate bond is subsequently formed the process of oxidation or sulfurization, which leads to the conjugation of the compound represented by Formula (321) with siRNA.

In some embodiments, the first functional group contains a protected hydroxy group. In some embodiments, the second functional group contains a group that can react with the solid phase support to produce a conjugating molecule containing the solid phase support. In some embodiments, the second functional group contains a carboxyl, carboxylate, or amidophosphite, as represented by Formula (C1), (C2), or (C3). When the second functional group contains a carboxyl or carboxylate, the compound represented by Formula (321) reacts with a hydroxy group or an amino group on a solid-phase support such as a resin through an esterification or amidation reaction to form a conjugating molecule containing the solid-phase support linked through a carboxyester bond. When the second functional group contains an amidophosphite functional group, the compound represented by Formula (321) can be bonded to a hydroxy group on a universal solid-phase support such as a resin and form, by oxidation, a conjugating molecule containing the solid-phase support linked through a phosphodiester bond. Then, starting from the above product bound to a solid-phase support, nucleoside monomers are sequentially coupled using a solid-phase amidophosphite synthesis method, thereby obtaining a sense strand or an antisense strand of siRNA linked to a conjugating group. During solid-phase amidophosphite synthesis, the first functional group is deprotected and then coupled to an amidophosphite group on a nucleoside monomer under coupling reaction conditions.

In some embodiments, the first functional group contains a hydroxy group or a protected hydroxy group; and the second functional group contains a carboxyester bonded solid support, an amide bonded solid support, or a phosphoester bonded solid support as represented by Formula (C1') or (C3'). In this case, starting from the compound represented by Formula (321), instead of a solid-phase support, nucleoside monomers are sequentially coupled using solid-phase amidophosphite synthesis, thereby obtaining a sense strand or an antisense strand of siRNA linked to a conjugating group.

In some embodiments, the carboxylate may be designated as -COO ^- M ⁺ , where M ⁺ is a cation such as one of a metal cation, an NH4 ⁺ ammonium cation, and an organic ammonium cation. In one embodiment, the metal ion may be an alkali metal ion such as K ⁺ or Na ⁺ . To increase solubility and facilitate the reaction, in some embodiments, the organic ammonium ion is a tertiary amine ammonium cation or a quaternary ammonium cation such as a triethylamine ammonium ion or an N,N-diisopropylethylamine ammonium ion. In some embodiments, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

In some embodiments, R ₄ contains a structure represented by Formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11') or (B12'):

where q ₁ is an integer from 1 to 4, q ₂ is an integer from 1 to 10, X is O or NH, M ⁺ is a cation, R _k is a hydroxy protecting group, TPP is a solid phase support, and denotes the site of covalent addition of a group. In some embodiments, q ₁ is 1 or 2. In some embodiments, q ₂ is an integer from 1 to 5. In some embodiments, R ₄ contains the structure represented by Formula (B9) or (B10). In some embodiments, R ₄ contains the structure represented by Formula (B11) or (B12).

In some embodiments, R _k is one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl). In some embodiments In an embodiment, R _k may be DMTr, i.e. 4,4'-dimethoxytrityl.

The definition of L ₁ is as described above.

In some embodiments, L ₁ is used to link the targeting group M ₁ to the N atom on the nitrogen backbone, which provides the siRNA conjugate represented by Formula (308) with liver targeting function. In some embodiments, L ₁ contains any of Formulas (A1) - (A26) or a combination thereof.

In accordance with the above description, those skilled in the art will readily understand that, compared with solid phase amidophosphite synthesis methods well known in the art, an siRNA conjugate in which the conjugating molecule is linked to any possible position of the nucleotide sequence can be prepared using the above first functional group and an optional second functional group. For example, a conjugating molecule is linked to the end of a nucleotide sequence or to both ends of a nucleotide sequence. Accordingly, unless otherwise indicated, in the following description, when referring to reactions such as “deprotection,” “coupling,” “capping,” “oxidation,” “sulfurization,” when referring to the preparation of an siRNA conjugate and/or conjugating molecule, it is intended that these reactions will also be applicable to the reaction conditions and agents used in solid phase amidophosphite nucleic acid synthesis processes well known in the art. Examples of reaction conditions and agents will be described in detail below.

In some embodiments, each S ₁ is independently M ₁ . In some embodiments, each S ₁ independently represents a group formed by protecting at least one active hydroxy group in M ₁ with hydroxy protecting groups. In some embodiments, each S ₁ independently represents a group formed by protecting all active hydroxy groups in M ₁ with hydroxy protecting groups. In some embodiments, any hydroxy protecting group known to those skilled in the art can be used to protect the active hydroxy group on M ₁ . In some embodiments, the protected hydroxy group is indicated by the formula YCOO-, wherein each Y is independently selected from the group consisting of C ₁ -C ₁₀ alkyl and C ₆ -C ₁₀ aryl, wherein C ₁ -C ₁₀ alkyl and C ₆ -C ₁₀ aryl are optional substituted with one or more substituents selected from the group consisting of halogen and C ₁ -C ₆ alkyl. In some embodiments, each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C ₁ -C ₆ alkylphenyl.

In some embodiments, each S ₁ is independently selected from the group consisting of Formulas A46-A54:

In some embodiments, S ₁ is Formula A49 or A50.

In some embodiments, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl. In some embodiments, Y is methyl.

As mentioned earlier, the method for producing the siRNA conjugate represented by Formula (308) further includes the following steps: synthesizing another siRNA strand (for example, when the siRNA sense strand associated with the conjugating molecule is synthesized in the above step, the method further includes synthesizing the siRNA antisense strand by the method solid-phase synthesis, and vice versa); separation of the sense chain and the antisense chain; and annealing. In particular, in the isolation step, the solid phase support associated with the nucleotide sequence and/or the conjugated molecule is cleaved off, and at the same time the necessary protecting group is removed (in this case, each S ₁ group in the compound represented by Formula (321) is converted to the corresponding targeting group M ₁ ), producing a sense strand (or antisense strand) of the siRNA associated with a conjugating molecule and a corresponding antisense strand (or sense strand). The sense strand and the antisense strand are annealed to form a double-stranded RNA structure, thereby obtaining the siRNA conjugate represented by Formula (308).

In some embodiments, the method of producing an siRNA conjugate represented by Formula (308) further includes the following steps: contacting the compound represented by Formula (321) with a first nucleoside monomer at the 3' end of the sense strand or antisense strand under coupling reaction conditions in the presence of a coupling agent, thereby binding the compound represented by Formula (321) to the first nucleotide in the sequence; sequential binding of nucleoside monomers in the 3' to 5' direction to synthesize the sense strand or antisense strand of siRNA in accordance with the desired type of nucleotides and the sequence of the sense strand or antisense strand, under solid-phase amidophosphite synthesis conditions; where the compound represented by Formula (321) is a compound in which R4 contains a first functional group and a second functional group, the first functional group contains a protected hydroxy group, and the second functional group contains a group represented by Formula (C1') or (C3') , and the compound represented by Formula (321) is deprotected before binding to the first nucleoside monomer; and the coupling of each nucleoside monomer involves a four-step reaction of deprotection, coupling, capping and oxidation or sulfurization, thereby producing a sense strand or an antisense nucleic acid strand linked to the conjugating molecule; sequential binding of nucleoside monomers in the 3' to 5' direction to synthesize the sense strand or antisense strand of a nucleic acid in accordance with the type of nucleotides and the sequence of the sense strand or antisense strand, under solid-phase amidophosphite synthesis conditions; wherein the binding of each nucleoside monomer involves a four-step reaction of deprotection, coupling, capping and oxidation or sulfurization; removal of protective groups and cleavage of solid-phase support; isolation and purification to obtain the sense strand and the antisense strand; and annealing.

In some embodiments, the method of producing the siRNA conjugate represented by Formula (308) further includes the following steps: sequentially linking nucleoside monomers in a 3' to 5' direction to synthesize the sense strand or antisense strand according to the type of nucleotides and the sequence of the sense strand or antisense strands in double-stranded siRNA; wherein the binding of each nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, and oxidation or sulfurization, thereby producing a solid-phase support-bound sense strand and a solid-phase support-linked antisense strand; bringing the compound represented by Formula (321) into contact with a sense strand bound to a solid phase support or an antisense strand bound to a solid phase support under coupling reaction conditions in the presence of a coupling agent, thereby binding the compound represented by Formula (321), with a sense strand or an antisense strand; wherein the compound represented by Formula (321) is a compound in which R ₄ contains an amidophosphite group as the first functional group; removal of protective groups and cleavage of solid-phase support; respectively, isolating and purifying to obtain the sense strand or antisense strand of siRNA; and annealing; where the sense strand or antisense strand of the siRNA is linked to a conjugating molecule.

In some embodiments, the P atom in Formula A59 is linked to the 3' end of the siRNA sense strand, and a method for preparing the siRNA conjugate represented by Formula (308) includes:

(1) removing a hydroxy protecting group R _k from a compound represented by Formula (321) (wherein the compound represented by Formula (321) is a compound in which R ₄ contains a first functional group and a second functional group, the first functional group contains a protected hydroxy group OR _k and the second functional group contains the structure represented by Formula (C1') or (C3')); and contacting the deprotected product with a nucleoside monomer to produce a nucleoside monomer bound to the solid phase support via a conjugating molecule under coupling reaction conditions in the presence of a coupling agent;

(2) synthesizing, starting from a nucleoside monomer bound to a solid-phase support through a conjugating molecule, the siRNA sense strand in the 3' to 5' direction using solid-phase amidophosphite synthesis;

(3) synthesis of the antisense strand of siRNA using solid-phase amidophosphite synthesis; And

(4) isolating the sense strand and the antisense strand of the siRNA and annealing them to obtain the siRNA conjugate represented by Formula (308).

In step (1), a method for removing a protecting group R _k from a compound represented by Formula (321) includes contacting the compound represented by Formula (321) with a deprotecting agent under deprotecting conditions. Deprotection conditions include a temperature of 0-50°C, and in some embodiments, 15-35°C, and a reaction duration of 30-300 seconds, and in some embodiments, 50-150 seconds. The deprotection agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, and in some embodiments, the deprotection agent is dichloroacetic acid. The molar ratio of the deprotecting agent to the compound represented by Formula (321) may be from 10:1 to 1000:1, and in some embodiments, from 50:1 to 500:1.

The coupling reaction conditions and the coupling reaction agent may be any conditions and agents suitable for the above coupling reaction. In some embodiments, the same conditions and agent may be used as in the coupling reaction in the solid phase synthesis method.

In some embodiments, coupling reaction conditions include a reaction temperature of 0-50°C, and in some embodiments, 15-35°C. The molar ratio of the compound represented by Formula (321) to nucleoside monomer may be from 1:1 to 1:50, and in some embodiments, from 1:2 to 1:5. The molar ratio of the compound represented by Formula (321) to the coupling agent may be from 1:1 to 1:50, and in some embodiments, from 1:3 to 1:10. The duration of the reaction may be 200-3000 seconds, and in some embodiments, 500-1500 seconds. The coupling agent may be selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, and in some embodiments is 5-ethylthio-1H-tetrazole. The organic solvent may be selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments is anhydrous acetonitrile. The amount of organic solvent may be 3-50 L/mol, and in some embodiments, 5-20 L/mol, relative to the compound represented by Formula (321).

In step (2), the sense strand SS of the siRNA conjugate is synthesized in the 3' to 5' direction by solid-phase amidophosphite nucleic acid synthesis, starting from the nucleoside monomer bound to the solid-phase support through the conjugating molecule obtained in the above steps. In this case, the conjugating molecule is bound to the 3' end of the resulting sense strand.

Other solid phase synthesis conditions in steps (2) and (3), including conditions for deprotecting the nucleoside monomer, type and amount of deprotecting agent, coupling reaction conditions, type and amount of coupling agent, capping reaction conditions, type and amount of capping agent , oxidation reaction conditions, type and amount of oxidizing agent, sulfurization reaction conditions, and type and amount of sulfurization agent include the use of agents, amounts, and conditions conventional in the art.

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

Deprotection conditions for the nucleoside monomer include a temperature of 0-50°C, and in some embodiments, 15-35°C, and a reaction time of 30-300 seconds, and in some embodiments, 50-150 seconds. The deprotection agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, and in some embodiments, the deprotection agent is dichloroacetic acid. The molar ratio of deprotectant to 4,4'-dimethoxytrityl protecting group on the solid phase support ranges from 2:1 to 100:1, and in some embodiments, from 3:1 to 50:1.

Coupling reaction conditions include a reaction temperature of 0-50°C, and in some embodiments, 15-35°C. The molar ratio of the nucleic acid sequence bound to the solid phase support to the nucleoside monomer is from 1:1 to 1:50, and in some embodiments from 1:5 to 1:15. The molar ratio of the nucleic acid sequence bound to the solid phase support to the coupling agent is from 1:1 to 1:100, and in some embodiments from 1:50 to 1:80. The duration of the reaction and the coupling reaction agent can be selected in the same manner as above.

Capping reaction conditions include a reaction temperature of 0-50°C, and in some embodiments, 15-35°C, and a reaction duration of 5-500 seconds, and in some embodiments, 10-100 seconds. The capping agent may be selected in the same manner as above. The molar ratio of the total amount of capping agent to the nucleic acid sequence bound to the solid phase support can be from 1:100 to 100:1, and in some embodiments is from 1:10 to 10:1. In the case where equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping agent, the molar ratio of acetic anhydride to N-methylimidazole to the nucleic acid sequence associated with the solid phase support may be from 1:1:10 to 10:10:1 , and in some embodiments is from 1:1:2 to 2:2:1.

Oxidation reaction conditions include a reaction temperature of 0-50°C, and in some embodiments, 15-35°C, and a reaction duration of 1-100 seconds, and in some embodiments, 5-50 seconds. In some embodiments, the oxidizing agent is iodine (in some embodiments, provided as iodine water). The molar ratio of oxidizing agent to nucleic acid sequence bound to the solid phase support in the coupling step can be from 1:1 to 100:1, and in some embodiments is from 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent in which the ratio of tetrahydrofuran:water:pyridine is from 3:1:1 to 1:1:3. The sulfurization reaction conditions include a reaction temperature of 0-50°C, and in some embodiments, 15-35°C, and a reaction duration of 50-2000 seconds, and in some embodiments, 100-1000 seconds. In some embodiments, the sulfurization agent is xanthan hydride. The molar ratio of the sulfurization agent to the nucleic acid sequence bound to the solid phase support in the coupling step is from 10:1 to 1000:1, and in some embodiments is from 10:1 to 500:1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent in which the acetonitrile:pyridine ratio is from 1:3 to 3:1.

The method further includes isolating the sense strand and antisense strand of the siRNA after binding of all nucleoside monomers and before annealing. Isolation methods are well known to those skilled in the art and typically involve cleavage of the synthesized nucleotide sequence from a solid phase support, removal of protecting groups on bases, phosphate groups and ligands, purification and desalting.

Conventional methods for cleavage and deprotection during siRNA synthesis can be used to cleave the synthesized nucleotide sequence from the solid phase support and remove protecting groups on bases, phosphate groups and ligands. For example, bringing the resulting nucleotide sequence associated with a solid-phase support into contact with a strong aqueous ammonia solution; during deprotection, the protecting group YCOO- on groups A46-A54 is converted to a hydroxy group, and thus the S ₁ groups are converted to the corresponding M ₁ group, obtaining the conjugate represented by Formula (308); where the strong aqueous ammonia solution may be an aqueous ammonia solution with a concentration of 25-30 wt.%. The amount of strong aqueous ammonia solution can be from 0.2 ml/μmol to 0.8 ml/μmol relative to the target siRNA.

When the synthesized nucleotide sequence has at least one 2'-TBDMS protecting group, the method further includes contacting the nucleotide sequence removed from the solid phase support with triethylamine trihydrofluoride to deprotect the 2'-TBDMS. In this case, the resulting siRNA target sequence contains the corresponding nucleoside having a free 2’-hydroxy group. The amount of pure triethylamine trihydrofluoride ranges from 0.4 ml/µmol to 1.0 ml/µmol relative to the target siRNA sequence. In this way, the siRNA conjugate represented by Formula (308) can be obtained.

Purification and desalting methods are well known to those skilled in the art. For example, nucleic acid purification can be accomplished using a preparative ion exchange chromatography purification column with NaBr or NaCl gradient elution; Once the product is collected and combined, desalting can be done using a reverse phase chromatography purification column.

The non-bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphate bond between nucleotides in the resulting siRNA conjugate represented by Formula (308) is substantially bound to a sodium ion, and the siRNA conjugate represented by Formula (308) is substantially present in the form of a sodium salt. Well-known ion exchange methods can be used, in which the sodium ion can be replaced with a hydrogen ion and/or other cations, providing other forms of siRNA conjugates represented by Formula (308). The cations are as described above.

During synthesis, the purity and molecular weight of the nucleic acid sequence can be determined at any time. For better control of the quality of the synthesis, such determination methods are well known to those skilled in the art. For example, nucleic acid purity can be determined using ion exchange chromatography, and molecular weight can be determined using liquid chromatography-mass spectrometry (LC-MS).

Annealing methods are also well known to those skilled in the art. For example, the synthesized sense strand (SS strand) and antisense strand (AS strand) can simply be mixed in equimolar ratio in water for injection, heated to 70-95°C, and then cooled at room temperature to form a double-stranded structure via hydrogen bonding. connections. In this way, the siRNA conjugate represented by Formula (308) can be obtained.

After preparing the conjugate, in some embodiments, the thus synthesized siRNA conjugate represented by Formula (308) can also be characterized by means such as molecular weight determination, methods such as liquid chromatography-mass spectrometry to confirm that the synthesized conjugate The siRNA is the target designed siRNA conjugate represented by Formula (308), and the sequence of the synthesized siRNA is the sequence of the siRNA that was required to be synthesized, for example, is one of the sequences listed in Tables 1a-1f.

The compound represented by Formula (321) can be produced by the following method, including: contacting the compound represented by Formula (313) with a cyclic anhydride in an organic solvent under an esterification reaction condition in the presence of a base and an esterification catalyst; and isolating the compound represented by Formula (321) by ion exchange:

Formula (313),

where the definitions and options n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L ₁ and S ₁ , respectively, are as described above;

R ₆ is the group providing R ₄ of Formula (321). In some embodiments, R ₆ contains the structure represented by Formula (A61):

,

(A61),

where R _i represents any group capable of bonding to an N atom on a nitrogen-containing backbone, bonding to R _k O and bonding to a free hydroxy group; and R _k represents a hydroxy protecting group. In this case, a compound represented by Formula (321) is obtained, wherein R ₄ contains a first functional group as a hydroxy protecting group and a second functional group containing a group represented by Formula (C1) or (C2).

The esterification reaction conditions include a reaction temperature of 0-100°C and a reaction time of 8-48 hours. In some embodiments, the esterification reaction conditions include a reaction temperature of 10-40°C and a reaction time of 20-30 hours.

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

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

The esterification catalyst may be any catalyst capable of catalyzing esterification, for example the catalyst may be 4-dimethylaminopyridine. The molar ratio of catalyst to compound represented by Formula (313) is from 1:1 to 10:1, and in some embodiments from 2:1 to 5:1.

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

Ion exchange has the function of converting the compound represented by Formula (321) into the target form of carboxylic acid or carboxylic acid salt, and ion exchange methods are well known to those skilled in the art. The above conjugating molecule in which the cation is M ⁺ can be prepared using a suitable ion exchange solution and ion exchange conditions, which are not described in detail herein. In some embodiments, a triethylamine phosphate solution is used in the ion exchange reaction, and the concentration of the triethylamine phosphate solution is 0.2-0.8 M. In some embodiments, the concentration of the triethylamine phosphate solution is 0.4-0.6 M. In some embodiments, the amount of triethylamine phosphate solution is 3-6 L/mol, and in a further embodiment 4-5 L/mol, relative to the compound represented by Formula (313).

The compound represented by Formula (321) can be isolated from the reaction mixture using any suitable isolation methods. In some embodiments, the compound represented by Formula (321) can be isolated by solvent removal by evaporation followed by chromatography, for example, using the following two isolation chromatographic conditions: (1) normal phase purification with a 200-300 mesh silica gel filter and gradient elution 1 wt. triethylamine in a mixture of dichloromethane:methanol in a ratio of 100:18 to 100:20; or (2) reverse phase purification with C18 and C8 reverse phase excipient and gradient elution with 0.1:1 to 1:0.1 methanol:acetonitrile. In some embodiments, the solvent can be immediately removed to obtain the crude product of the compound represented by Formula (321), which can be immediately used in subsequent reactions.

In some embodiments, the method of producing the compound represented by Formula (321) further includes: contacting the product obtained from the above ion exchange reaction with a solid phase support containing amino groups or hydroxy groups in an organic solvent under condensation reaction conditions in the presence of a condensing agent , condensation catalyst and tertiary amine. In this case, a compound represented by Formula (321) is obtained, wherein R ₄ contains a first functional group containing a hydroxy protecting group and a second functional group having the structure represented by Formula (C1').

A solid phase support is one of the supports used in solid phase siRNA synthesis, some of which are well known to those skilled in the art. For example, the solid phase support may be selected from solid phase supports containing an active hydroxy or amino functional group. In some embodiments, the solid phase support is an amino resin or a hydroxy resin. In some embodiments, the amino resin or hydroxy resin has the following parameters: a particle size of 100-400 mesh and a specific amino or hydroxy group content of 0.2-0.5 mmol/g. The ratio of the compound represented by Formula (321) to the solid support is 10-400 μmol of compound per gram of solid support (μmol/g). In some embodiments, the ratio of compound of Formula (321) to solid phase support is 50-200 µmol/g.

The organic solvent may be any suitable solvent or mixed solvents known to those skilled in the art. In some embodiments, the organic solvent includes one or more of acetonitrile, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The amount of organic solvent may be 20-200 L/mol, and in some embodiments, 50-100 L/mol, relative to the compound represented by Formula (321).

In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT), and/or O-benzotriazole -1-yl-tetramethyluronium hexafluorophosphate. In some embodiments, the condensing agent is O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by Formula (321) is from 1:1 to 20:1, and in some embodiments from 1:1 to 5:1.

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

In some embodiments, a method for preparing the compound represented by Formula (321) further includes: contacting the resulting condensation product with a capping agent and an acylation catalyst in an organic solvent under a capping reaction condition, and isolating the compound represented by Formula (321). The capping reaction is used to remove any reactive functional groups that are completely unreactive to avoid the formation of by-products in subsequent reactions. Capping reaction conditions include a reaction temperature of 0-50°C, and in some embodiments, 15-35°C, and a reaction duration of 1-10 hours, and in some embodiments, 3-6 hours. The capping agent may be a capping agent used in solid-phase siRNA synthesis, and the capping agent used in solid-phase siRNA synthesis is well known to those skilled in the art.

In some embodiments, the capping agent consists of capping agent 1 (cap1) and capping agent 2 (cap2). Cap1 is N-methylimidazole, and in some embodiments is provided as a mixed solution of N-methylimidazole in pyridine/acetonitrile, wherein the volume ratio of pyridine to acetonitrile is from 1:10 to 1:1, and in some embodiments from 1:3 up to 1:1. In some embodiments, the ratio of the total volume of pyridine and acetonitrile to the volume of N-methylimidazole is from 1:1 to 10:1, and in some embodiments from 3:1 to 7:1. Capping agent 2 is acetic anhydride. In some embodiments, capping agent 2 is provided as a solution of acetic anhydride in acetonitrile, where the volume ratio of acetic anhydride to acetonitrile is from 1:1 to 1:10, and in some embodiments from 1:2 to 1:6.

In some embodiments, the ratio of the volume of the mixed solution of N-methylimidazole in pyridine/acetonitrile to the weight of the compound represented by Formula (321) is from 5 ml/g to 50 ml/g, and in some embodiments from 15 ml/g to 30 ml /G. The ratio of the volume of a solution of acetic anhydride in acetonitrile to the weight of the compound represented by Formula (321) is from 0.5 ml/g to 10 ml/g, and in some embodiments, from 1 ml/g to 5 ml/g.

In some embodiments, the capping agent contains equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent includes one or more of acetonitrile, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The amount of organic solvent may be 10-50 L/mol, and in some embodiments, 5-30 L/mol, relative to the compound represented by Formula (321).

In some embodiments, the acylation catalyst may be selected from any catalyst that can be used for esterification or amide condensation, such as alkaline heterocyclic compounds. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The weight ratio of catalyst to compound represented by Formula (321) may be from 0.001:1 to 1:1, and in some embodiments, from 0.01:1 to 0.1:1.

In some embodiments, the compound represented by Formula (321) can be isolated from the reaction mixture using any suitable isolation methods. In some embodiments, the compound represented by Formula (321) can be prepared by thorough washing with an organic solvent and filtration to remove unreacted reagents, excess capping agent, and other impurities, where the organic solvent is selected from acetonitrile, dichloromethane, or methanol. In some embodiments, the organic solvent is acetonitrile.

In some embodiments, producing the conjugating molecule represented by Formula (321) involves contacting the compound represented by Formula (313) with diamidophosphite in an organic solvent under coupling reaction conditions in the presence of a coupling agent, and isolating the compound represented by Formula (321) . In this case, a compound represented by Formula (321) is obtained, wherein R ₄ contains a first functional group containing a hydroxy protecting group and a second functional group having the structure represented by Formula (C3).

In some embodiments, the coupling reaction conditions include a reaction temperature of 0-50°C, for example, 15-35°C. The molar ratio of the compound represented by Formula (313) to diamidophosphite may be from 1:1 to 1:50, such as 1:5 to 1:15. The molar ratio of the compound represented by Formula (313) to the coupling agent may be from 1:1 to 1:100, for example from 1:50 to 80. The duration of the reaction can be 200-3000 seconds, for example 500-1500 seconds. The diamidophosphite may be, for example, a bis(diisopropylamino)(2-cyanoethoxy)phosphine, which may be commercially available or synthesized according to methods well known in the art. The coupling agent is selected from one or more of 1H-tetrazole, 5-ethylthio-1H-tetrazole and 5-benzylthio-1H-tetrazole, for example 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent, and the organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF (dimethylformamide) and anhydrous dichloromethane, such as anhydrous acetonitrile. The amount of the organic solvent may be 3-50 L/mol, such as 5-20 L/mol, relative to the compound represented by Formula (313). In a coupling reaction, the hydroxy group in the compound represented by Formula (313) reacts with diamidophosphite to form an amidophosphite group. In some embodiments, the solvent can be immediately removed to obtain the crude product of the compound represented by Formula (321), which can be immediately used in subsequent reactions.

In some embodiments, a method for preparing the compound represented by Formula (321) further comprises: contacting the isolated product with a solid phase support containing hydroxy groups in an organic solvent under coupling reaction conditions in the presence of a coupling agent, followed by capping, oxidation and isolation, with obtaining the compound represented by Formula (321). In this case, a compound represented by Formula (321) is obtained, wherein R ₄ contains a first functional group containing a hydroxy protecting group and a second functional group having the structure represented by Formula (C3').

In some embodiments, the solid phase support is a well known solid phase support in the field of solid phase nucleic acid synthesis, such as a commercially available unprotected universal solid phase support (NittoPhase®HL UnyLinker™ 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, represented by Formula B80):

(B80).

The deprotection reaction is well known in the art. In some embodiments, deprotection conditions include a temperature of 0-50°C, such as 15-35°C, and a reaction duration of 30-300 seconds, such as 50-150 seconds. The deprotection agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid and monochloroacetic acid. In some embodiments, the deprotection agent is dichloroacetic acid. The molar ratio of the deprotecting agent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the solid phase support can be from 2:1 to 100:1, for example from 3:1 to 50:1. With this deprotection, reactive hydroxy groups are produced on the surface of the solid phase support to facilitate the subsequent coupling reaction.

The coupling reaction conditions and the coupling reaction agent may be selected as above. During the coupling reaction, the free hydroxy groups formed in the deprotection reaction react with the amidophosphite groups to form a phosphite ester linkage.

In some embodiments, the capping reaction conditions include a reaction temperature of 0-50°C, such as 15-35°C, and a reaction duration of 5-500 seconds, such as 10-100 seconds. The capping reaction is carried out in the presence of a capping agent. The capping agent and its amount are selected as above.

The oxidation reaction conditions may include a temperature of 0-50°C, for example 15-35°C, and a reaction time of 1-100 seconds, for example 5-50 seconds. The oxidizing agent may be, for example, iodine (in some embodiments, provided as iodine water). In some embodiments, the molar ratio of the oxidizing agent to the nucleic acid sequence associated with the solid phase support is from 1:1 to 100:1, for example, from 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent in which the ratio of tetrahydrofuran:water:pyridine is from 3:1:1 to 1:1:3.

In some embodiments, R ₆ is a group represented by Formula B7 or B8:

where the definitions of q ₂ and R _k correspond to those described above.

In this case, the compound represented by Formula (313) can be produced by the following method: bringing the compound represented by Formula (314) into contact with the compound represented by Formula (A-1) or the compound represented by Formula (A-2 ), in an organic solvent under amidation reaction conditions in the presence of an amide condensation agent and a tertiary amine, and isolating:

where the definitions and options n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , L ₁ , S ₁ , q ₂ and R _k respectively are as described higher.

The amidation reaction conditions may include a reaction temperature of 0-100°C and a reaction time of 1-48 hours. In some embodiments, the amidation reaction conditions include a reaction temperature of 10-40°C and a reaction time of 2-16 hours.

In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the alcohol solvent is one or more of methanol, ethanol, and propanol, and in some embodiments is ethanol. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and/or methyl tert-butyl ether. In some embodiments, the haloalkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of organic solvent is 3-50 L/mol, and in additional embodiments, 3-20 L/mol, relative to the compound represented by Formula (314).

In some embodiments, the amide condensation agent is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one, 4-(4,6-dimethoxytriazine-2 -yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate, and in additional embodiments is 3-(diethoxyphosphoryloxy)- 1,2,3-benzotriazin-4(3H)-one. The molar ratio of the amide condensation agent to the compound represented by Formula (314) may be from 1:1 to 10:1, and in some embodiments, from 2.5:1 to 5:1.

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

The compounds represented by Formula (A-1) and Formula (A-2) can be prepared by any suitable methods. For example, when R _k represents a DMTr group, the compound represented by Formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl. Likewise, the compound represented by Formula (A-2) can be prepared by contacting 3-amino-1,2-propanediol with a cyclic anhydride and then reacting with DMTrCl, wherein the cyclic anhydride may contain 4 to 13 carbon atoms, and in some embodiments, 4-8 carbon atoms. Those skilled in the art will readily appreciate that the cyclic anhydrides selected correspond to the various q ₂ values in the compound represented by Formula (A-2). For example, if the cyclic anhydride is succinic anhydride, q ₂ =1; if the cyclic anhydride is glutaric anhydride, q ₂ =2, and so on.

In some embodiments, the compound represented by Formula (313) can also be prepared by sequentially reacting the compound represented by Formula (314) with a cyclic anhydride, 3-amino-1,2-propanediol and DMTrCl. Those skilled in the art will readily understand that these variations will not affect the structure and function of the compound represented by Formula (313), and these variations can be easily implemented by those skilled in the art based on the above methods.

Likewise, the compound represented by Formula (313) can be isolated from the reaction mixture using any suitable isolation methods. In some embodiments, the compound represented by Formula (313) can be isolated by solvent removal by evaporation followed by chromatography, for example, using the following two isolation chromatography conditions: (1) normal phase purification with a 200-300 mesh silica gel filter and gradient elution with a mixture of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide in a ratio of 1:1:1:0.5 to 1:1:1:0.6; and (2) reverse phase purification with C18 and C8 reverse phase excipients and gradient elution with 0.1:1 to 1:0.1 methanol:acetonitrile. In some embodiments, the solvent can be immediately removed to obtain the crude product of the compound represented by Formula (313), which can be immediately used in subsequent reactions.

In some embodiments, the compound represented by Formula (314) can be prepared by the following method, comprising: contacting the compound represented by Formula (320) with the compound represented by Formula (316) in an organic solvent under condensation reaction conditions in the presence of an amide condensation agent and a tertiary amine, and the release of:

Formula (316)

Formula (320),

where the definitions and options n1, n3, m1, m2, m3, R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ respectively are as described above.

The compound represented by Formula (316) may be as described in J. Am. Chem. Soc. 2014, 136, 16958-16961, or the compound represented by Formula (316), can be prepared by those skilled in the art using various methods. For example, certain compounds represented by Formula (316) can be prepared in accordance with the methods disclosed in Example 1 of US Pat. No. 8,106,022 B2, incorporated herein by reference in its entirety.

In some embodiments, the condensation reaction conditions include a reaction temperature of 0-100°C and a reaction duration of 0.1-24 hours. In some embodiments, the condensation reaction conditions include a reaction temperature of 10-40°C and a reaction duration of 0.5-16 hours.

Considering the structure of the target compound represented by Formula (314), the molar ratio of the compound represented by Formula (316) to the compound represented by Formula (320) should be determined based on the sum of n1 and n3 in Formula (320). In some embodiments, for example, when n1+n3=3, to ensure complete reaction and no excess, the molar ratio of the compound represented by Formula (316) to the compound represented by Formula (320) may be from 3:1 to 3 .5:1, and in some embodiments is between 3.01:1 and 3.15:1.

In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy solvent, an ether solvent, a haloalkane solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran. In some embodiments, the ether solvent is diethyl ether and/or methyl tert-butyl ether. In some embodiments, the haloalkane solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The amount of organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol, relative to the compound represented by Formula (320).

In some embodiments, the amide condensation agent is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT), O-benzotriazol-1-yl -tetramethyluronium hexafluorophosphate, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, or 1-hydroxybenzotriazole, and in additional embodiments is a mixture of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazole -1-yl-hydroxytripyrrolidinophosphonium hexafluorophosphate (PyBop) and 1-hydroxybenzotriazole are contained in equimolar quantities. The molar ratio of the total amount of amide condensation agent to the compound represented by Formula (316) may be from 1:1 to 3:1, and in some embodiments is from 1.05:1 to 1.5:1.

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

Likewise, the compound represented by Formula (314) can be isolated from the reaction mixture using any suitable isolation methods. In some embodiments, the compound represented by Formula (314) is isolated by solvent removal by evaporation followed by chromatography, for example, using the following two isolation chromatography conditions: (1) normal phase 200-300 mesh silica gel filter purification and gradient elution with a mixture of dichloromethane:methanol in a ratio of 100:5 to 100:7; and (2) reverse phase purification with C18 and C8 reverse phase excipients and gradient elution with 0.1:1 to 1:0.1 methanol:acetonitrile. In some embodiments, the solvent is immediately removed to obtain the crude product of the compound represented by Formula (314), which can be immediately used in subsequent reactions.

The compound represented by Formula (320) may be commercially available or prepared by those skilled in the art using known methods. For example, in the case where m1=m2=m3=3, n1=1, n3=2, and R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are all H, the compound represented by Formula ( 320), available commercially from Alfa Aesar Inc.

The siRNA conjugate of the present invention may also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventional in the art. Detailed information is contained in the above description of the pharmaceutical compositions according to the present invention.

Use of siRNA, pharmaceutical composition and siRNA conjugate according to the present invention

Some embodiments of the present invention provide the use of an siRNA and/or a pharmaceutical composition and/or a siRNA conjugate of the present invention for the production of a medicament for the treatment and/or prevention of myasthenia gravis.

According to some embodiments of the present invention, there is provided a method of preventing and/or treating myasthenia gravis, comprising administering an effective amount of siRNA and/or a pharmaceutical composition and/or siRNA conjugate of the present invention to a subject in need.

The purpose of preventing and/or treating myasthenia gravis can be achieved based on the RNA interference mechanism by administering the active ingredients of the siRNA according to the present invention to a subject in need. Thus, the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present invention can be used for the prevention and/or treatment of myasthenia gravis or for the production of a medicament for the prevention and/or treatment of myasthenia gravis.

In connection with the present invention, the term "administration" refers to the delivery of siRNA, pharmaceutical composition and/or siRNA conjugate according to the present invention into the body of a subject using a method or route that at least partially localizes siRNA, pharmaceutical composition and/or siRNA conjugate according to the present invention in the desired location to obtain the desired effect. Suitable routes of administration for the methods of the present invention include local administration and systemic administration. In general, local administration results in the delivery of a greater amount of siRNA conjugate to a specific site compared to the systemic circulation of the subject, while systemic administration results in delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present invention substantially into the systemic circulation of the subject. Whereas the present invention may provide agents for the prevention and/or treatment of myasthenia gravis, some embodiments employ a route of administration capable of delivering drugs to the liver.

Administration to a subject may be by any suitable route known in the art, including, but not limited to, the oral or parenteral route, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, intratracheal administration (aerosol), inhalation administration , intranasal administration, rectal administration and local administration (including buccal administration and sublingual administration). The frequency of administration may be one or more times per day, weekly, once every two weeks, once every three weeks, monthly, once every two months, once every three months, once every six months, or annually.

The dose of the siRNA, pharmaceutical composition or second siRNA conjugate according to the present invention may be a conventional dose in the art, and the dose may be determined according to various parameters, particularly the age, body weight and gender of the subject. Toxicity and efficacy can be measured in cell cultures or experimental animals using standard pharmaceutical procedures, for example, by determining LD ₅₀ (lethal dose that causes death of 50% of the population), ED ₅₀ (dose that, when quantitatively responded, can cause 50% of the population). maximum intensity of response, and with a qualitative response provides a positive response in 50% of test subjects) or IC ₅₀ (concentration of inhibitor/drug required to suppress the quantitative response by half). Dose ranges for humans can be derived from data obtained from cell culture assays and animal studies.

When the siRNA, pharmaceutical composition or siRNA conjugate of the present invention is administered to, for example, male or female C57BL/6J mice aged 6-12 weeks and weighing 18-25 g, and calculated based on the amount of siRNA: (i) for the siRNA conjugate the dosage of siRNA contained therein may be 0.001-100 mg/kg body weight, and in additional embodiments is 0.01-50 mg/kg body weight, and in some embodiments is 0.05-20 mg/kg body weight, in some other embodiments, the implementation is 0.1-15 mg/kg body weight, and in some other embodiments, the implementation of the present invention is 0.1-10 mg/kg body weight; and (ii) for a pharmaceutical composition consisting of siRNA and a pharmaceutically acceptable carrier, the dosage of the siRNA contained therein may be 0.001-50 mg/kg body weight, in some embodiments, 0.01-10 mg/kg body weight, in some in embodiments, is 0.05-5 mg/kg body weight, and in some embodiments, is 0.1-3 mg/kg body weight.

In some embodiments, the present invention provides a method of inhibiting C5 gene expression in a hepatocyte. Said method involves bringing an effective amount of siRNA and/or pharmaceutical composition and/or siRNA conjugate according to the present invention into contact with said hepatocyte, introducing siRNA and/or pharmaceutical composition and/or siRNA conjugate according to the present invention into said hepatocyte and achieving the purpose of inhibiting the expression of the C5 gene in the specified hepatocyte through the mechanism of RNA interference. The hepatocyte may be selected from SMMC-7721, HepG2, Huh7 and other hepatoma cell lines or isolated primary hepatocytes. In some embodiments, the cells are HepG2 hepatoma cells.

When inhibiting C5 expression in a cell using the method of the present invention, the amount of siRNA in the proposed modified siRNA, pharmaceutical composition and/or siRNA conjugate is generally an amount sufficient to reduce expression of the target gene and achieve an extracellular concentration of 1 pM to 1 µM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or 0.05 nM to approximately 5 nM at the surface of the target cell. The amount required to achieve this local concentration will vary depending on various factors, including the method of delivery, the site of delivery, the number of cellular layers between the site of delivery and the target cells or tissues, the route of delivery (local or systemic), etc. The concentration at the site of delivery may be significantly higher than the concentration on the surface of the target cells or tissues.

Kit

The present invention provides a kit containing an effective amount of at least one of a modified siRNA, a pharmaceutical composition, and a siRNA conjugate of the present invention.

In some embodiments, a kit disclosed herein may provide the modified siRNA in a single container. In some embodiments, the kit of the present invention may contain a container in which pharmaceutically acceptable excipients are provided. In some embodiments, the kit may further contain additional ingredients, such as stabilizers or preservatives. In some embodiments, a kit of the present invention may contain at least one additional therapeutic agent in a container different from the container in which the modified siRNA of the present invention is provided. In some embodiments, the kit may contain instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or adjuvants or other ingredients (if any).

In the kit of the present invention, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvants, as well as the modified siRNA, pharmaceutical composition and/or siRNA conjugate, and/or pharmaceutically acceptable adjuvants may be provided in any form, for example, liquid form, dry form or lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvants, as well as the pharmaceutical composition and/or siRNA conjugate and optional pharmaceutically acceptable adjuvants are substantially pure and/or sterile. In some embodiments, the kit of the present invention may provide sterile water.

Hereinafter, the present invention will be further described by way of examples, but is not limited thereto in any respect.

Examples

Unless otherwise noted, all agents and culture media used in the following examples are commercially available, and all procedures used, such as nucleic acid electrophoresis and real-time PCR, were performed in accordance with the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

Lipofectamine™ 2000 (Invitrogen) was used as a transfection reagent when transfecting cells with siRNA and siRNA conjugate to the C5 gene synthesized in the present invention, or siRNA and siRNA conjugate as a negative control, and the specific process followed the instructions provided by the manufacturer.

Unless otherwise noted, all reactant ratios given below are calculated as ratios by volume (v/v).

All experimental data are expressed as , and data analysis was performed using Graphpad prism 5.0 statistical analysis software.

Preparation example 1. Preparation of conjugate 1

In this preparation example, conjugate 1 (ie L10-siC5a1M1SP) was synthesized. The conjugated siRNA in the conjugate has the sense strand and antisense strand sequences corresponding to conjugate 1 from Table 3.

(1-1) Synthesis of compound L-10

Compound L-10 was synthesized according to the following method:

(1-1-1) Synthesis of GAL-5 (terminal conjugating molecule)

(1-1-1a) Synthesis of GAL-2

100.0 g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No. 1772-03-8, purchased from Ningbo Hongxiang Bio-Chem Co., Ltd., 463.8 mmol) was dissolved in 1000 ml of anhydrous pyridine, to which 540 ml of acetic anhydride (purchased from Enox Inc., 5565.6 mmol) was added in an ice water bath to react with stirring at room temperature for 1.5 hours. The resulting reaction solution was poured into 10 L of ice water and subjected to vacuum filtration under reduced pressure. The residue was washed with 2 L of ice water, and then acetonitrile/toluene mixed solvent (v/v ratio = 1:1) was added until completely dissolved. The solvent was removed by evaporation to obtain 130.0 g of product GAL-2 as a white solid.

(1-1-1b) Synthesis of GAL-3

GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was dissolved in 213 ml of anhydrous 1,2-dichloroethane, to which was added 24.0 g of trimethylsilyl trifluoromethanesulfonate (TMSOTf, CAS No. 27607-77-8, purchased from Macklin Inc., 108.0 mmol) in an ice water bath and under a protective nitrogen atmosphere to react at room temperature overnight.

400 ml of dichloromethane was added to the reaction solution to dilute it, filtered through diatomaceous earth, and then 1 L of saturated aqueous sodium bicarbonate was added and stirred until homogeneous. The organic phase was isolated. The remaining aqueous phase was extracted twice, each time with 300 ml of dichloroethane, and all organic phases were combined and washed with 300 ml of saturated aqueous sodium bicarbonate solution and 300 ml of saturated brine, respectively. The organic phase obtained from washing was isolated and dried using anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain 26.9 g of product GAL-3 as a light yellow viscous syrup.

(1-1-1c) Synthesis of GAL-4

GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane, 30 g of dry molecular sieve powder was added 4 followed by 9.0 g of 5-hexen-1-ol (CAS No. 821-41-0, purchased from Adamas-beta Inc., 89.9 mmol), and stirred at room temperature for 30 minutes. 9.08 ml TMSOTf (40.9 mmol) was added in an ice bath and under a protective nitrogen atmosphere to react with stirring at room temperature overnight. Molecular Sieve Powder 4 removed by filtration. 300 ml of dichloroethane was added to the filtrate for dilution, filtered through diatomaceous earth, and then 500 ml of saturated aqueous sodium bicarbonate was added and stirred for 10 minutes to wash. The organic phase was isolated. The aqueous phase was extracted once with 300 ml of dichloroethane. All organic phases were combined and washed with 300 ml saturated aqueous sodium bicarbonate solution and 300 ml saturated brine, respectively. The organic phase obtained from washing was isolated and dried using anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain 41.3 g of GAL-4 product as a yellow syrup, which was immediately used in the next oxidation reaction without purification.

(1-1-1d) Synthesis of GAL-5

GAL-4 (14.9 g, 34.7 mmol) prepared according to the method described in step (1-1 1c) was dissolved in a mixed solvent of 77 ml dichloromethane and 77 ml acetonitrile, added 103 ml deionized water and 29.7 g of sodium periodate (CAS No. 7790-28-5, purchased from Aladdin Inc., 138.8 mmol), respectively, and stirred in an ice bath for 10 minutes. Ruthenium trichloride (CAS No. 14898-67-0, purchased from Energy Chemical, 238 mg, 1.145 mmol) was added to react at room temperature overnight. The resulting reaction solution was diluted by adding 300 ml of water with stirring and adjusted to a pH of approximately 7.5 by adding saturated sodium bicarbonate. The organic phase was isolated and discarded. The aqueous phase was extracted three times, each time with 200 ml of dichloromethane, and the organic phase resulting from the extraction was discarded. The aqueous phase obtained from the extraction was adjusted to a pH of approximately 3 with citric acid powder, extracted three times, each time with 200 ml of dichloromethane, and the resulting organic phases were combined and dried with anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain 6.85 g of product GAL-5 as a white foamy solid. ^1H 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).

(1-1-2) Synthesis of L-8:

J-0 (9.886 g, 52.5 mmol, purchased from AlfaAesar) and GAL-5 (72.819 g, 162.75 mmol, prepared by combining products from several batches) obtained in step (1-1-1) were dissolved in 525 ml of dichloromethane, diizopropidylamine was added (DIEA, 44.782 g, 346.50 mmol), benzotriazole-1-il-octirolidinophosphonia hexaphotosphate (Pybop, 90.158 g, 173.25 mmol) and hydroxybenzorisol (Hobt, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23, 23 410 g, 173.25 mmol) react at room temperature for 4 hours, and then add 20 ml saturated sodium bicarbonate and 200 ml saturated brine for washing. The aqueous phase was extracted twice, each time with 100 ml of dichloromethane, and the resulting organic phases were combined and dried with anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure to obtain the crude product. The crude product was purified using a normal phase silica gel column (200-300 mesh). 10 wt.% triethylamine was added to the column to neutralize the acidity of silica gel, balanced with 1 wt. triethylamine and eluted using a gradient elution mode with a mixture of dichloromethane:methanol in a ratio of 100:30 to 100:40. The eluate was collected and the solvent was removed by evaporation under reduced pressure to obtain 38.8 g of pure product L-8. ^1H NMR (400 MHz, DMSO) δ 7.84 (d, J = 9.0 Hz, 3H), 7.27 - 7.23 (m, 1H), 7.13 - 7.18 (m, 1H ), 5.22 (d, J = 3.1 Hz, 3H), 4.97 (dd, J = 11.3, 3.1 Hz, 3H), 4.48 (d, J = 8.4 Hz , 3H), 4.09 - 3.98 (m, 9H), 3.88 (dd, J = 19.3, 9.3 Hz, 3H), 3.75 - 3.66 (m, 3H), 3.44 - 3.38 (m, 3H), 3.17 - 3.30 (m, 4H), 3.10 - 2.97 (m, 4H), 2.35 - 2.20 (m, 6H ), 2.15 - 2.08 (m, 9H), 2.07 - 1.98 (m, 13H), 1.94 - 1.87 (m, 9H), 1.81 - 1.74 (m , 9H), 1.65 - 1.42 (m, 18H). MS m/z: C ₈₅ H ₁₁₉ N ₇ O ₃₀ , [M+H] ⁺ , calculated: 1477.59, measured: 1477.23.

(1-1-3a) Synthesis A-1

DMTrCl (4,4'-dimethoxytrityl chloride, 101.65 g, 300 mmol) was dissolved in 1000 ml anhydrous pyridine and calcium DL-glycerate hydrate (28.63 g, 100 mmol) was added to react at 45°C for 20 hours. The reaction solution was filtered. The residue was washed with 200 ml DCM and the filtrate was concentrated to dryness under reduced pressure. The residue was redissolved in 500 ml of dichloromethane and washed twice, each time with 200 ml of 0.5 M triethylamine phosphate (pH=7-8). The isolated aqueous phase was extracted twice, each time with 200 ml of dichloromethane. All organic phases were combined, dried with anhydrous sodium sulfate and filtered. The solvent was removed by evaporation under reduced pressure and the residue was purified using a normal phase silica gel column (200-300 mesh) eluted using a 1:1:1 petroleum ether:ethyl acetate:dichloromethane:methanol gradient. :0.35 to 1:1:1:0.55. The eluate was collected and the solvent was removed by evaporation under reduced pressure. The residue was redissolved in 600 ml of dichloromethane and washed once with 200 ml of 0.5 M triethylamine phosphate. The isolated aqueous phase was extracted once with 200 ml of dichloromethane. All organic phases were combined, dried with anhydrous sodium sulfate and filtered. The solvent was removed by evaporation under reduced pressure and overnight under reduced pressure using a vacuum oil pump to obtain 50.7 g of product A-1 as a white solid. ^1H NMR (400 MHz, DMSO-d6) δ 7.46 (ddd, J = 6.5, 2.3, 1.1 Hz, 1H), 7.40 - 7.28 (m, 7H), 6 .89 - 6.81 (m, 4H), 4.84 (d, J = 5.0 Hz, 1H), 4.36 - 4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J = 12.4, 7.0 Hz, 1H), 3.67 (dd, J = 12.3, 7.0 Hz, 1H), 2.52 (q, J = 6, 3 Hz, 6H), 1.03 (t, J = 6.3 Hz, 9H). MS m/z: C ₂₄ H ₂₃ O ₆ , [MH] ^- , calculated: 407.15, measured: 406.92.

(1-1-3b) Synthesis of L-7

L-8 (40 g, 27.09 mmol, prepared by combining products from several batches) obtained from step (1-1-2), and A-1 (41.418 g, 81.27 mmol) obtained from step (1 -1-3a) were mixed and dissolved in 271 ml of dichloromethane, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT, 24.318 g, 81.37 mmol) was added and then diisopropylethylamine (21.007 g, 162.54 mmol) to react with stirring at 25°C for 1.5 hours. The organic phase was washed with 800 ml of saturated sodium bicarbonate solution. The isolated aqueous phase was extracted three times, each time with 50 ml of dichloromethane. The organic phase was washed with 150 ml of saturated brine and the aqueous phase was extracted once with 50 ml of dichloromethane. The resulting organic phases were combined and dried using anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure and the residue was foam dried using an oil vacuum pump overnight to obtain the crude product. The crude product was subjected to column purification. The column was filled with 2 kg of normal phase silica gel (200-300 mesh), 200 ml of triethylamine was added to neutralize the acidity of the silica gel, equilibrated with petroleum ether containing 1 wt.% triethylamine, and eluted in a gradient elution mode with a mixture of petroleum ether: ethyl acetate: dichloromethane: N ,N-dimethylformamide in a ratio from 1:1:1:0.5 to 1:1:1:0.6. The eluate was collected and the solvent was removed by evaporation under reduced pressure to obtain 40.4 g of pure product L-7. ^1H NMR (400 MHz, DMSO) δ 7.90 - 7.78 (m, 4H), 7.75 - 7.64 (m, 1H), 7.38 - 7.18 (m, 9H), 6 .91 - 6.83 (m, 4H), 5.25 - 5.10 (m, 4H), 4.97 (dd, J = 11.2, 3.2 Hz, 3H), 4.48 - 4 .30 (m, 4H), 4.02 (s, 9H), 3.93 - 3.84 (m, 3H), 3.76 - 3.66 (m, 9H), 3.45 - 3.35 (m, 3H), 3.24 - 2.98 (m, 10H), 2.30 - 2.20 (m, 2H), 2.11 - 1.88 (m, 31H), 1.80 - 1 .40 (m, 28H). MS m/z: C ₉₀ H ₁₂₈ N ₇ O ₃₅ , [M-DMTr] ⁺ , calculated: 1564.65, measured: 1564.88.

(1-1-4) Synthesis of L-9

L-7 (40 g, 21.4247 mmol) obtained from step (1-1-3b), succinic anhydride (4.288 g, 42.8494 mmol) and 4-dimethylaminopyridine (DMAP, 5.235 g, 42.8494 mmol) mixed and dissolved in 215 ml of dichloromethane, then diisopropylethylamine (DIPEA, 13.845 g, 107.1235 mmol) was added and stirred at 25°C for 24 hours. The reaction solution was washed with 800 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted three times, each time with 5 ml of dichloromethane. All organic phases were combined and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was subjected to column purification. The column was filled with 1 kg of normal phase silica gel (200-300 mesh), 1 wt.% triethylamine was added to neutralize the acidity of the silica gel, equilibrated with dichloromethane and eluted in a gradient elution mode with a mixture of dichloromethane (containing 1 wt. triethylamine):methanol in a ratio of 100:18 to 100:20. The eluate was collected and the solvent was evaporated under reduced pressure to obtain 31.0 g of pure L-9 conjugating molecule product. ^1H NMR (400 MHz, DMSO) δ 8.58 (d, J = 4.2 Hz, 1H), 7.94 - 7.82 (m, 3H), 7.41 - 7.29 (m, 5H ), 7.22 (d, J = 8.1 Hz, 5H), 6.89 (d, J = 8.3 Hz, 4H), 5.49 - 5.37 (m, 1H), 5.21 (d, J = 3.0 Hz, 3H), 4.97 (d, J = 11.1 Hz, 3H), 4.49 (d, J = 8.2 Hz, 3H), 4.02 (s , 9H), 3.88 (dd, J = 19.4, 9.4 Hz, 3H), 3.77 - 3.65 (m, 9H), 3.50 - 3.39 (m, 6H), 3.11 - 2.90 (m, 5H), 2.61 - 2.54 (m, 4H), 2.47 - 2.41 (m, 2H), 2.26 - 2.17 (m, 2H ), 2.15 - 1.95 (m, 22H), 1.92 - 1.84 (m, 9H), 1.80 - 1.70 (m, 10H), 1.65 - 1.35 (m , 17H), 1.31 - 1.19 (m, 4H), 0.96 (t, J = 7.1 Hz, 9H). MS m/z: _{C94H132N7O38 ,} [ _M -DMTr] ⁺ , calculated: 1664.72, measured: _1665.03 .

(1-1-5) Synthesis of compound L-10:

At this stage, compound L-10 was prepared by coupling the conjugating molecule L-9 to a solid phase support.

The conjugating molecule L-9 (22.751 g, 11 mmol) obtained in step (1-1-4), O-benzotriazol-1-yl-tetramethyluronium hexafluorophosphate (HBTU, 6.257 g, 16.5 mmol) and diisopropylethylamine (DIEA, 2.843 g, 22 mmol) were mixed, dissolved in 900 ml of acetonitrile and stirred at room temperature for 5 minutes. Aminomethyl resin (88 g, 100-200 mesh, amino content: 400 μmol/g, purchased from Tianjin Nankai HECHENG S&T Co., Ltd.) was added to the reaction solution. The reaction was carried out on a shaker at 25°C and 150 rpm for 18 hours, followed by filtration. The residue was washed twice, each time with 300 ml DCM, and washed three times, each time with 300 ml acetonitrile, and dried for 18 hours using a vacuum oil pump. The capping reaction was then carried out by adding starting materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) according to the ratio shown in Table 2. The reaction was carried out on a shaker at 25°C and 150 rpm for 5 hours. The reaction solution was filtered. The residue was washed three times, each time with 300 ml of acetonitrile, the solvent was evaporated to dryness and the mixture was dried overnight under reduced pressure using an oil vacuum pump to obtain 102 g of compound L-10 (i.e. the conjugating molecule L-9 bound to solid-phase support), with a content of 90.8 µmol/g.

Table 2. Ratio of starting materials in the capping reaction

Starting materials Quantity Qualification Serial No. Manufacturer CapA 1980 ml - - - CapB 220 ml - - - DMAP 1,100 g Clean for analysis I1422139 Aladdin Acetonitrile 220 ml Spectral pure O15161001 CINC (Shanghai) Co.,Ltd

In the table above, CapA and CapB represent solutions of capping agents. CapA is a 20 vol.% solution of N-methylimidazole in a mixture of pyridine/acetonitrile, where the volume ratio of pyridine to acetonitrile is 3:5. CapB is a 20 vol.% solution of acetic anhydride in acetonitrile.

(1-2) Synthesis of the sense strand of conjugate 1

Nucleoside monomers were sequentially conjugated in the 3' to 5' direction according to the sequence of nucleotides in the sense strand by solid-phase phosphoamidite synthesis, cycles starting with compound L-10 obtained in the above step to synthesize the sense strand SS conjugate 1 from Table 3. Binding of each nucleoside monomer involved a four-step reaction of deprotection, coupling, capping, and oxidation or sulfurization. When two nucleotides were linked via a phosphoester, a four-step reaction of deprotection, coupling, capping and oxidation was carried out during the binding of the next nucleoside monomer. When two nucleotides were linked via a thiophosphate, a four-step deprotection, coupling, capping, and sulfurization reaction was performed while the next nucleoside monomer was bound. The synthesis conditions were set as follows.

Nucleoside monomers were provided in 0.1 M acetonitrile solution. The deprotection reaction conditions were identical in all steps, i.e., temperature 25°C, reaction time 70 seconds, dichloroacetic acid solution in dichloromethane (3% v/v) as deprotecting agent and molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on a solid-phase support, equal to 5:1.

The coupling reaction conditions were identical in all stages and included a temperature of 25°C, a molar ratio of the nucleic acid sequence bound to the solid phase support to nucleoside monomers of 1:10, a molar ratio of the nucleic acid sequence bound to the solid phase support to the coupling agent , equal to 1:65, reaction time of 600 seconds and 0.5 M solution of 5-ethylthio-1H-tetrazole (ETT) in acetonitrile as the coupling agent.

The capping reaction conditions were identical for all stages and included a temperature of 25°C and a reaction duration of 15 seconds. The capping agent was a mixed solution of Cap A and Cap B in a 1:1 molar ratio, and the molar ratio of the capping agent to the nucleic acid sequence bound to the solid phase support was 1:1:1 (anhydride:N-methylimidazole:nucleic acid sequence, associated with the solid-phase substrate).

The oxidation reaction conditions were identical for all stages and included a temperature of 25°C, a reaction time of 15 seconds, and 0.05 M iodine water as the oxidizing agent. The molar ratio of iodine to nucleic acid sequence bound to the solid phase support during the coupling step was 30:1. The reaction was carried out in a mixed solvent in which the ratio of tetrahydrofuran:water:pyridine was 3:1:1.

The sulfurization reaction conditions were identical in all stages and included a temperature of 25°C, a reaction time of 300 seconds, and xanthan hydride as the sulfurization agent. The molar ratio of sulfurization agent to nucleic acid sequence bound to the solid phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent in which the ratio of acetonitrile: pyridine was 1:1.

After binding of the last nucleoside monomer, the nucleic acid sequence bound to the solid phase support was cleaved, deprotected, purified and desalted, and then lyophilized to produce the sense strand, where

The conditions for cleavage and deprotection were as follows: adding the synthesized nucleotide sequence bound to the support into a 25 wt.% aqueous ammonia solution to react for 16 hours at 55°C, where the amount of aqueous ammonia solution was 0.5 ml/μmol; filtration to remove the support and concentration of the supernatant in vacuo to dryness.

Purification and desalting conditions were as follows: nucleic acid purification using a preparative ion exchange chromatography column (Source 15Q) with NaCl gradient elution. Specifically, eluent A: 20 mM sodium phosphate (pH = 8.1), solvent: water/acetonitrile = 9:1 (v/v); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), solvent: water/acetonitrile = 9:1 (v/v); elution gradient: eluent A:eluent B = 100:0 to 50:50. The eluate was collected, pooled and desalted using a reverse phase chromatography purification column. Specific conditions included using a Sephadex column (packing: Sephadex-G25) for desalting and deionized water for elution.

The detection method was as follows: determination of the purity of the above-described sense chain by ion exchange chromatography (IOC-HPLC); and molecular weight analysis by liquid chromatography-mass spectrometry (LC-MS). The measured value was consistent with the calculated value, indicating the synthesis of the sense strand SS conjugated to the conjugating molecule L-9 at the 3′ end.

(1-3) Synthesis of the antisense chain of conjugate 1

The antisense chain AS of conjugate 1 from Table 3 was synthesized by starting cycles using a universal solid-phase support (NittoPhase ^® HL Solid-Phase Supports loaded with UnyLinker™, Kinovate Life Sciences Inc.) according to the solid-phase amidophosphite synthesis method. The solid-phase synthesis method used the same conditions for deprotection, coupling, capping, oxidation or sulfurization reactions, and the same conditions for elimination and deprotection, purification and desalting as for the synthesis of the sense chain. The difference was that the antisense strand contained a 5'-phosphoric acid at the first nucleotide at the 5' end. Therefore, in the process of preparing the antisense chain according to the solid-phase amidophosphite synthesis method, after binding the last nucleoside monomer of the antisense chain, the CPR-I monomer (Suzhou GenePharma, Part No. Cat. No. 13-2601-XX) was linked to the 5' end of the antisense chain with formation of a 5'-phosphoester modification through four steps of deprotection, coupling, capping and oxidation.

(CPR-I)

For this coupling, the same conditions for the deprotection, coupling, capping and oxidation reactions, and the same conditions for elimination and deprotection, purification and desalting, were used as for the synthesis of the sense chain. The residue was lyophilized to obtain the antisense chain. The purity of the antisense chain was determined by ion exchange chromatography (IOC-HPLC), and the molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). The measured value was consistent with the calculated value, indicating the synthesis of the antisense strand AS having the target sequence.

(1-4) Synthesis of conjugate 1

The sense strand and the antisense strand were respectively dissolved in water for injection to obtain a 40 mg/ml solution, mixed in an equimolar ratio, heated at 50°C for 15 minutes, and then cooled at room temperature, resulting in an annealed product, and then lyophilized to obtain lyophilized powder. The conjugate was diluted to a concentration of 0.2 mg/mL with ultrapure water (prepared using a Milli-Q ultrapure water unit with a resistivity of 18.2 MΩ*cm (25°C)). Molecular weight was measured by liquid chromatography-mass spectrometry (LC-MS, instrument purchased from Waters Corp., model: LCT Premier). Since the measured value was consistent with the calculated value, it was confirmed that the synthesized conjugate 1 was the target designed double-stranded nucleic acid sequence with the conjugating molecule L-9. The structure of conjugate 1 corresponded to that presented in Formula (403), the sequence of the conjugated siRNA corresponded to the sequence of conjugate 1 (i.e., L10-siC5a1M1SP), as shown in Table 3.

Preparation example 2. Preparation of conjugates 2-8

Conjugates 2-6, presented in Table 3, were synthesized in the same way as in Preparation Example 1, and the molecular weights were determined. The difference was that the sense and antisense sequences used in the synthesis were the sequences presented in Table 3, corresponding to the sense and antisense strands of the conjugated siRNAs in conjugate 2, conjugate 3, conjugate 4, conjugate 5, or conjugate 6. respectively, resulting in conjugates 2-6, respectively.

Conjugates 7-8 shown in Table 3 were synthesized in the same manner as in Preparation Example 1, and the molecular weights were determined. The difference was that the sense and antisense sequences used in the synthesis were the sequences presented in Table 3, corresponding to the sense and antisense strands of the conjugated siRNAs in conjugate 7 or conjugate 8, respectively, with the antisense strands of conjugate 7 and conjugate 8 do not have a 5'-phosphoric acid on the first nucleotides at their 5' ends compared to the antisense strand of conjugate 1. Therefore, in the process of preparing antisense strands according to the solid-phase amidophosphite synthesis method, there is no need to bind the CPR-I monomer after binding the latter nucleoside monomer of the antisense chain, obtaining conjugates 7-8, respectively.

Table 3 lists the conjugate numbers and siRNA sequence compositions.

Table 3. siRNA conjugates

Conjugate No. Sequence recording direction 5’-3’ SEQ ID NO: Conjugate 1 L10-siC5a1M1SP Semantic chain CmsUmsUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 361 Antisense strand PUmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm 362 Conjugate 2 L10-siC5b1M1SP Semantic chain CmsUmsAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 363 Antisense strand PUmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm 364 Conjugate 3 L10-siC5c1M1SP Semantic chain GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 365 Antisense strand PUmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm 366 Conjugate 4 L10-siC5d1M1SP Semantic chain AmsGmsAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 367 Antisense strand PUmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm 368 Conjugate 5 L10-siC5e1M1SP Semantic chain CmsCmsAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 369 Antisense strand PUmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm 370 Conjugate 6 L10-siC5f1M1SP Semantic chain CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 371 Antisense strand PUmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm 372 Conjugate 7 L10-siC5c1M1S Semantic chain GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 377 Antisense strand UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm 378 Conjugate 8 L10-siC5d1M1S Semantic chain AmsGmsAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 379 Antisense strand UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm 380

Preparation example 3. Synthesis of siRNA sequence

siRNA 1 shown in Table 4a was synthesized in the same way as in Preparation Example 1, with the difference that:

1) for the sense strand, cycles were started using a universal solid phase support (NittoPhase ^® HL solid phase supports loaded with UnyLinker™, Kinovate Life Sciences Inc.); And

2) for the antisense strand, compared to the sequence of the antisense strand of siRNA conjugated in conjugate 1, the first nucleotide at the 5' end of siRNA 1 did not contain 5' phosphoric acid. Therefore, in the process of preparing antisense chains according to the solid-phase amidophosphite synthesis method, it was not necessary to bind the CPR-I monomer after binding the last nucleoside monomer of the antisense chain.

Thus, siRNA 1 was obtained.

miRNA 2-6 were synthesized in the same way as miRNA 1, with the difference that the sequences of the sense strands and antisense strands of the siRNA used in the synthesis were the sequences presented in Table 4, corresponding to the sense strands and antisense strands of siRNA 2, siRNA 3, miRNA 4, miRNA 5, or miRNA 6, respectively, resulting in miRNAs 2–6, respectively.

Table 4a lists the miRNA numbers and miRNA sequence compositions.

Table 4a. miRNA sequences

siRNA No. Sequence recording direction 5’-3’ SEQ ID NO: miRNA 1 siC5a1M1S Semantic chain CmsUmsUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm 373 Antisense strand UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm 374 miRNA 2 siC5b1M1S Semantic chain CmsUmsAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm 375 Antisense strand UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm 376 miRNA 3 siC5c1M1S Semantic chain GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 377 Antisense strand UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm 378 miRNA 4 siC5d1M1S Semantic chain AmsGmsAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm 379 Antisense strand UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm 380 miRNA 5 siC5e1M1S Semantic chain CmsCmsAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm 381 Antisense strand UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm 382 miRNA 6 siC5f1M1S Semantic chain CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 383 Antisense strand UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm 384

Preparation example 4. Synthesis of Cy5-labeled siRNA and Cy5-labeled siRNA conjugates

(4-1) Synthesis of Cy5-conjugate 1 and Cy5-conjugate 2

Cy5 conjugate 1 shown in Table 4b was synthesized in the same manner as in Preparation Example 1, and the molecular weight was determined. The difference was that the sense strand and antisense strand sequences used in the synthesis were the sequences shown in Table 4b, corresponding to the sense strand and antisense strand of the siRNA conjugated in Cy5-conjugate 1, wherein (1): Cy5 fluorescent group was covalently linked to the 5' end of the siRNA sense strand conjugated in Cy5-conjugate 1. Therefore, in the process of preparing the sense strand according to the solid-phase amidophosphite synthesis method described in step (1-2) of Preparation Example 1, after binding the last nucleoside of the sense strand monomer, it was necessary to link the Cy5 amidophosphite derivative monomer (purchased from Shanghai HonGene Biotech, Part No. OP-057) to the 5' end of the sense strand through a four-step reaction of deprotection, coupling, capping, and oxidation; and (2) the first nucleotide at the 5′ end of the siRNA conjugated in Cy5-conjugate 1 did not contain 5′-phosphoric acid; therefore, in the process of preparing antisense chains according to the solid-phase amidophosphite synthesis method described in step (1-3) of Preparation Example 1, it was not necessary to bind the CPR-I monomer after binding the last nucleoside monomer of the antisense chain.

(monomer of amidophosphite derivative Cy5)

In the process of binding the Cy5 amidophosphite derivative monomer to the 5' end of the sense strand, the same deprotection, coupling, capping and oxidation reaction conditions were used as described for the synthesis of the sense strand in step (1-2) of Preparation Example 1, and the differences were that: 1) the duration of the deprotection reaction was increased to 300 seconds; and 2) the duration of the Cy5 coupling reaction was increased to 900 seconds.

Then, the conditions for cleavage and deprotection of the sense strand were as follows: adding the synthesized nucleotide sequence bound to the support into the AMA solution (mixed solution of 40 wt.% aqueous methylamine solution and 25 wt.% aqueous ammonia solution in a 1:1 volume ratio) , where the amount of AMA solution was 0.5 ml/μmol, reacting for 2 hours in a water bath at 25°C, removing the remaining support by filtration and concentrating the supernatant to dryness in vacuum. The conditions for purification and desalting of the sense strand were the same as those for the synthesis of the sense strand in step (1-2) in Preparation Example 1. The residue was then lyophilized to obtain the sense strand of Cy5-conjugate 1.

Thus, Cy5-conjugate 1 was obtained, and the Cy5 fluorescent group was covalently linked to the 5' end of the siRNA sense strand in the siRNA conjugate, which had the sense strand and antisense strand sequences shown in Table 4b, corresponding to Cy5-conjugate 1.

Cy5-conjugate 2 was prepared in the same manner as Cy5-conjugate 1, and the molecular weight was determined. The difference was that the sense strand and antisense strand sequences used in the synthesis were the sequences shown in Table 4b, corresponding to the sense strand and antisense strand of the siRNA conjugated in Cy5-conjugate 2, respectively, resulting in the Cy5-conjugate 2.

(4-2) Synthesis of Cy5-siRNA 1 and Cy5-siRNA 2

Cy5-siRNA 1 was prepared in the same manner as Cy5-conjugate 1, and the molecular weight was determined. The difference was that the runs were started using a universal solid-phase support (NittoPhase ^® HL Solid-Phase Supports loaded with UnyLinker™, Kinovate Life Sciences Inc.) and resulted in Cy5-miRNA 1.

Cy5-siRNA 2 was prepared in the same manner as Cy5-siRNA 1, and the molecular weight was determined. The difference was that the sequences of the sense strands and antisense strands used in the synthesis were the sequences shown in Table 4b, corresponding to the sense strand and antisense strand of Cy5-siRNA, respectively, resulting in Cy5-siRNA 2.

Table 4b lists the siRNA numbers and sequences of Cy5-conjugate 1, Cy5-conjugate 2, Cy5-siRNA 1, and Cy5-siRNA 2.

Table 4b. Sequences of siRNAs in conjugates of Cy5-labeled siRNAs and Cy5-labeled siRNAs

siRNA or conjugate No. Sequence recording direction 5’-3’ SEQ ID NO: Cy5-miRNA 1 Cy5-siC5c1M1S Semantic chain GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 377 Antisense strand UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm 378 Cy5-miRNA 2 Cy5-siC5f1M1S Semantic chain CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 383 Antisense strand UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm 384 Cy5-conjugate 1 Cy5-L10-siC5c1M1S Semantic chain GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm 377 Antisense strand UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm 378 Cy5-conjugate 2 Cy5-L10-siC5f1M1S Semantic chain CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm 383 Antisense strand UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm 384

Experimental Example 1: Determination of IC ₅₀ of siRNA conjugate against C5 mRNA in HepG2 cells

HepG2 cells (purchased from Nanjing COBIOER Biotechnology Co., Ltd.) were cultured in complete H-DMEM (from HyClone) containing 10% fetal bovine serum (FBS, from RMBIO) and 0.2 vol% penicillin-streptomycin. (from HyClone), at 37°C in an incubator containing 5% CO ₂ /95% air.

HepG2 cells were seeded in a 24-well plate at a rate of 7x10 ⁴ cells/well. After 16 hours, when the cells reached 70-80% confluency, complete H-DMEM medium was aspirated from the culture wells and 500 μl of Opti-MEM medium (from GIBCO) was added to each well to continue culture for 1.5 hours.

From each of conjugate 1, conjugate 2, conjugate 3, conjugate 4, conjugate 5, conjugate 6, conjugate 7 and conjugate 8, working solutions of the conjugate were prepared with 7 different concentrations, including 20 μM, 5 μM, 1.25 μM, 0.313 μM, 0.0781 µM, 0.0195 µM and 0.0049 µM (in terms of siRNA), in DEPC (diethyl pyrocarbonate) treated water, respectively.

For each conjugate, solution A1-A7 was prepared, and each solution contained 3 μl of conjugate working solution with 7 concentrations as shown above and 50 μl of Opti-MEM medium.

Solution B was prepared separately for each conjugate, and each solution B contained 1 μl of Lipofectamine™ 2000 and 50 μl of Opti-MEM medium.

For each conjugate, one portion of solution B was mixed sequentially with one portion of solution A1-A7, and then incubated for 20 minutes at room temperature to obtain transfection complexes X1-X7. Two portions were prepared for each transfection complex.

One portion of solution B was mixed with 50 μl of Opti-MEM medium and incubated for 20 minutes at room temperature to obtain the X8 transfection complex. Four portions of the transfection complex were prepared.

Transfection complexes X1-X7 corresponding to each of the conjugates were added to the above culture wells for culturing HepG2 cells at a rate of 100 μl/well and mixed evenly to obtain transfection mixtures with final concentrations (in terms of siRNA) of 100 nM, 25 nM, 6. 25 nM, 1.56 nM, 0.391 nM, 0.098 nM and 0.024 nM, respectively. Two transfection complexes X1-X7 at the same concentration were added to two different culture wells to obtain transfection mixtures containing conjugates that were labeled test groups 1-7.

One portion of X8 transfection complex was added to the remaining four culture wells at a rate of 100 μl/well to obtain a transfection mixture without conjugate, which was labeled as the control group.

After incubation of the transfection mixture containing the conjugate and the transfection mixture without the conjugate with cells in culture wells for 4 hours, 1 ml of complete H-DMEM containing 20% FBS was added to each well. The 24-well plate was placed in an incubator containing 5% CO ₂ /95% air for 24 hours.

Then, RNAVzol (purchased from Vigorous Biotechnology Beijing Co., Ltd., Item No. N002) was used to extract total RNA from the cells in each well according to the total RNA extraction process steps in the instructions for use.

1 μg of total RNA was taken as a template for reverse transcription for cells in each well and reagents from the Goldenstar™ RT6 cDNA Synthesis Kit (purchased from Beijing Tsingke Biotechnology Co., Ltd., item no. TSK301M) were used. using Goldenstar™ Oligo (dT) ₁₇ as the primer, and 20 μl of the reverse transcription reaction system was prepared according to the reverse transcription process steps in the kit instructions to reverse the total cell RNA from each well. The reverse transcription reaction conditions were as follows: the reverse transcription reaction system was incubated at 50°C for 50 minutes, then incubated at 85°C for 5 minutes, and finally incubated at 4°C for 30 seconds. After the reaction, 80 μL of DEPC-treated water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

For each reverse transcription reaction system, 5 μL of solution containing cDNA was taken as a qPCR template, and 20 μL of qPCR reaction system was prepared using the reagent from the NovoStart ^® SYBR qPCR SuperMix Plus kit (purchased from Novoprotein Science and Technology Co., Ltd ., art. No. E096-01B), where the sequences of PCR primers for amplification of the target gene C5 and the internal control GAPDH gene are presented in Table 5, and the final concentration of each primer was 0.25 μM. The qPCR reaction system was placed on ABI's StepOnePlus real-time PCR instrument and a three-step amplification process was performed. The amplification procedure included pre-denaturation at 95°C for 10 minutes, then denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 30 seconds. After repeating the above denaturation, annealing, and elongation processes 40 times, product W was obtained containing the amplified C5 target gene and the internal control GAPDH gene. Product W was then alternately incubated at 95°C for 15 seconds, 60°C for 1 minute and 95°C for 15 seconds. The melting curves of the C5 target gene and the internal control GAPDH gene in product W were recorded by quantitative real-time PCR with fluorescence detection, and the Ct (cycle threshold) values for the C5 target gene and the internal control GAPDH gene were obtained.

Table 5. Primer sequences for detection

Gene Direct primer (direction 5’-3’) Reverse primer (direction 5’-3’) Human C5 ATCAGGCCAGGGAAGGTTAC(SEQ ID NO: 385) TCGGGATGAAGGAACCATGT(SEQ ID NO: 386) Human GAPDH GGTCGGAGTCAACGGATTT
(SEQ ID NO: 387) CCAGCATCGCCCCACTTGA
(SEQ ID NO: 388)

The comparative Ct estimation method (ΔΔCt) was used to relative quantify C5 target gene expression in each test group and control group. The calculation method was as follows:

ΔCt (test group) = Ct (target gene of the test group) - Ct (internal control gene of the test group)

ΔCt (control group) = Ct (target gene of control group) - Ct (internal control gene of control group)

ΔΔCt (test group) = ΔCt (test group) - ΔCt (control group)

ΔΔCt (control group) = ΔCt (control group) - ΔCt (control group mean)

where ΔCt (control group mean) was the arithmetic mean of the ΔCt (control group) value of each of the four culture wells of the control group. Thus, each culture well of each test group corresponded to one ΔΔCt (test group), and each culture well of the control group corresponded to one ΔΔCt (control group).

The level of C5 mRNA expression in the test group was normalized relative to the control group, where the level of C5 mRNA expression in the control group was taken as 100%.

Relative expression level of C5 mRNA in test group = 2^(-ΔΔCt(test group)) × 100%.

Based on the relative expression levels of C5 mRNA in HepG2 cells transfected with different conjugates (conjugates 1-8), dose-response curves were generated for conjugates 1-8 as shown in FIG. 1A-1H, by approximation with a three-parameter model “log(inhibitor) vs. response" using Graphpad 5.0 software, where the logarithm (lg nM) of the final concentration of siRNA conjugate was plotted on the x-axis, and the relative expression level (%) of C5 mRNA was plotted on the y-axis, and each point represented the average value of the relative expression level of C5 mRNA in two culture wells of the test group compared to the control group.

The IC ₅₀ of each conjugate against C5 mRNA was calculated based on the function corresponding to the fitted dose-response curve, where the function was as follows:

Where:

Y represents the relative expression level of C5 mRNA of the test group,

X is the logarithm of the final siRNA conjugate concentration,

Bot represents the Y value in the lower plateau region,

Top represents the Y value at the top plateau region, and

X' is the value of X where Y is the median value between the bottom and top of the asymptote, and HillSlope is the slope of the curve at X', which in this case is defined as -1.

Based on the dose-response curve and the corresponding function, the corresponding _X50 value when Y=50% was determined, and the _IC50 value of each conjugate was calculated to be 10^ _X50 (nM).

The IC ₅₀ value for C5 mRNA and the curve fit R ² value for each conjugate are summarized in Table 6.

Table 6. IC ₅₀ value for C5 mRNA and R ² value of the fitted dose-response curve for the siRNA conjugate

Conjugate No. IC ₅₀ R ² Conjugate 1 L10-siC5a1M1SP 9.688 nM 0.9513 Conjugate 2 L10-siC5b1M1SP 9.566 nM 0.9609 Conjugate 3 L10-siC5c1M1SP 2.861 nM 0.9958 Conjugate 4 L10-siC5d1M1SP 1.494 nM 0.9418 Conjugate 5 L10-siC5e1M1SP 4.077 nM 0.9942 Conjugate 6 L10-siC5f1M1SP 3.795 nM 0.9817 Conjugate 7 L10-siC5c1M1S 3.023 nM 0.9975 Conjugate 8 L10-siC5d1M1S 1.629 nM 0.9961

From the results in FIG. 1A-1H and Table 6 above, it can be seen that the siRNA conjugate of the present invention has high inhibitory activity in HepG2 hepatoma cells in vitro, and the IC ₅₀ ranges from 1.494 nM to 9.688 nM.

Experimental Example 2: Distribution of siRNA conjugates to various organs of C57 mice

A 1× PBS solution was used to dissolve Cy5-siRNA 1, Cy5-conjugate 1, Cy5-siRNA 2, or Cy5-conjugate 2, respectively, to obtain a solution of 0.6 mg/ml (in terms of siRNA).

Ten 6-7 week old female C57BL/6J mice (purchased from Beijing Charles River Laboratory Animal Technology Co., Ltd., called C57 mice for short) were randomly divided into groups, two mice in each group, and subcutaneously injected accordingly 1 × PBS and a solution of Cy5-siRNA 1, Cy5-conjugate 1, Cy5-siRNA 2 or Cy5-conjugate 2 obtained above. The administered doses for all animals were calculated according to their body weight, and the administration volume was 5 ml/kg body weight. In terms of siRNA, the administered dose for each animal was 3 mg/kg body weight.

At 2 hours, 6 hours, 24 hours, and 48 hours after administration, mice in each group were placed in the IVIS Lumina Series III small animal in vivo optical imaging system. Mice were inhaled with isoflurane anesthesia and placed under anesthesia for in vivo imaging, belly up, in a small animal in vivo optical imaging system to dynamically detect Cy5 fluorescence signals and monitor the distribution of Cy5-labeled siRNAs or Cy5-labeled conjugates in live animals. Only mice injected with Cy5-labeled conjugates had significantly increased fluorescent signals in their liver region, and mice injected with Cy5-labeled siRNA or 1× PBS did not have any fluorescent signal in their liver region.

After 48 hours, all mice were sacrificed, and five organs from each mouse, including the heart, lungs, liver, spleen, and kidneys, were removed for fluorescence imaging on the IVIS Lumina Series III. One animal was selected from each group of mice and its above-mentioned five organs were arranged alternately in the longitudinal direction. Organs from mice treated with 1× PBS or Cy5-siRNA 1 or Cy5-conjugate 1 were photographed in the same field of view, and the results are shown in FIG. 2A. Organs of mice injected with 1× PBS and Cy5-siRNA 2 or Cy5-conjugate 2 were photographed in the same field of view, and the results are shown in FIG. 2B.

From FIG. 2A-2B, it can be seen that only Cy5-conjugate 1 and Cy5-conjugate 2 are able to accumulate in large quantities in the liver, and Cy5-conjugate 1 and Cy5-conjugate 2 accumulate in a small amount in the kidney, but do not accumulate in other organs, which indicates that the conjugate of the present invention is capable of efficiently delivering siRNA directly to the liver. Moreover, in combination with the results in FIG. 1G (showing that conjugate 7 has superior inhibitory activity in vitro) and FIG. 1F (demonstrating that conjugate 6 has higher inhibitory activity in vitro), this suggests that the conjugate of the present invention is capable of specifically inhibiting target gene expression in the liver.

Some embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above-described embodiments. Various simple changes to the technical solution according to the present invention can be made within the scope of the technical concept of the present invention, and these simple changes are within the scope of the present invention.

In addition, it should be noted that all of the specific technical features described in the above embodiments may be combined in any suitable manner, provided that this does not lead to conflict. To avoid unnecessary repetition, the various possible combinations will not be further described in the present specification.

In addition, various embodiments of the present invention may also be implemented in any combination, provided that they do not conflict with the essence of the present invention, and such combinations should also be considered part of the disclosure of the present invention.

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LIST OF SEQUENCES

<110> SUZHOU RIBO LIFE SCIENCE CO., LTD.

<120> NUCLEIC ACID, PHARMACEUTICAL COMPOSITION AND CONJUGATE,

METHOD OF OBTAINING AND APPLICATION

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<150>CN201910440575.8

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<210> 8

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<222> (1)..(1)

<221> misc_feature

<223> n represents Z4, Z4 represents a nucleotide,

complementary to Z3, and Z3 is selected from A, U, G or C

<400> 8

nuugucugua ugaaugaaga gaa

23

<210> 9

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 9

cuucauucau acagacaaa

19

<210> 10

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 10

uuugucugua ugaaugaaga g

21

<210> 11

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 11

cucuucauucauacagacaaa

21

<210> 12

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 12

uuugucugua ugaaugaaga gaa

23

<210> 13

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 13

cuucauucau acagacaaa

19

<210> 14

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 14

uuugucugua ugaaugaaga g

21

<210> 15

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 15

cuucauucau acagacaaa

19

<210> 16

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 16

uuugucugua ugaaugaaga g

21

<210> 17

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 17

cuucauucau acagacaaa

19

<210> 18

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 18

uuugucugua ugaaugaaga g

21

<210> 19

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 19

cucuucauucauacagacaaa

21

<210> 20

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 20

uuugucugua ugaaugaaga gaa

23

<210> 21

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 21

cucuucauucauacagacaaa

21

<210> 22

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 22

uuugucugua ugaaugaaga gaa

23

<210> 23

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 23

cucuucauucauacagacaaa

21

<210> 24

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 24

uuugucugua ugaaugaaga gaa

23

<210> 25

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 25

cuucauucau acagacaaa

19

<210> 26

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 26

uuugucugua ugaaugaaga g

21

<210> 27

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 27

cuucauucau acagacaaa

19

<210> 28

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 28

uuugucugua ugaaugaaga g

21

<210> 29

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 29

cuucauucau acagacaaa

19

<210> 30

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 30

uuugucugua ugaaugaaga g

21

<210> 31

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 31

cucuucauucauacagacaaa

21

<210> 32

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 32

uuugucugua ugaaugaaga gaa

23

<210> 33

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 33

cucuucauucauacagacaaa

21

<210> 34

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 34

uuugucugua ugaaugaaga gaa

23

<210> 35

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 35

cucuucauucauacagacaaa

21

<210> 36

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 36

uuugucugua ugaaugaaga gaa

23

<210> 37

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 37

cuucauucau acagacaaa

19

<210> 38

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 38

uuugucugua ugaaugaaga g

21

<210> 39

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 39

cuucauucau acagacaaa

19

<210> 40

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 40

uuugucugua ugaaugaaga g

21

<210> 41

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 41

cuucauucau acagacaaa

19

<210> 42

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 42

uuugucugua ugaaugaaga g

21

<210> 43

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 43

cucuucauucauacagacaaa

21

<210> 44

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 44

uuugucugua ugaaugaaga gaa

23

<210> 45

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 45

cucuucauucauacagacaaa

21

<210> 46

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 46

uuugucugua ugaaugaaga gaa

23

<210> 47

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 47

cucuucauucauacagacaaa

21

<210> 48

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 48

uuugucugua ugaaugaaga gaa

23

<210> 49

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 49

cuucauucau acagacaaa

19

<210> 50

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 50

uuugucugua ugaaugaaga g

21

<210> 51

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 51

cuucauucau acagacaaa

19

<210> 52

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 52

uuugucugua ugaaugaaga g

21

<210> 53

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 53

cuucauucau acagacaaa

19

<210> 54

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 54

uuugucugua ugaaugaaga g

21

<210> 55

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 55

cucuucauucauacagacaaa

21

<210> 56

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 56

uuugucugua ugaaugaaga gaa

23

<210> 57

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 57

cucuucauucauacagacaaa

21

<210> 58

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 58

uuugucugua ugaaugaaga gaa

23

<210> 59

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 59

cucuucauucauacagacaaa

21

<210> 60

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 60

uuugucugua ugaaugaaga gaa

23

<210> 61

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223>N is Z5, and Z5 is A

<400> 61

cuacaguuua gaagauuun

19

<210> 62

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223>n is Z6, and Z6 is U

<400> 62

naaaucuucu aaacuguag

19

<210> 63

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n represents Z7, and Z7 is selected from A, U, G or C

<400> 63

cuacaguuua gaagauuun

19

<210> 64

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z8, Z8 represents a nucleotide,

complementary to Z8, and Z7 selected from A, U, G or C

<400> 64

naaaucuucu aaacuguag

19

<210> 65

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n represents Z7, and Z7 is selected from A, U, G or C

<400> 65

cuacaguuua gaagauuun

19

<210> 66

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z8, Z8 represents a nucleotide,

complementary to Z8, and Z7 selected from A, U, G or C

<400> 66

naaaucuucu aaacuguagu a

21

<210> 67

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (21)..(21)

<223> n represents Z7, and Z7 is selected from A, U, G or C

<400> 67

uacuacaguu uagaagauuu n

21

<210> 68

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z8, Z8 represents a nucleotide,

complementary to Z8, and Z7 selected from A, U, G or C

<400> 68

naaaucuucu aaacuguagu aug

23

<210> 69

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 69

cuacaguuua gaagauuua

19

<210> 70

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 70

uaaaucuucu aaacuguagu a

21

<210> 71

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 71

uacuacaguu uagaagauuu a

21

<210> 72

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 72

uaaaucuucu aaacuguagu aug

23

<210> 73

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 73

cuacaguuua gaagauuua

19

<210> 74

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 74

uaaaucuucu aaacuguagu a

21

<210> 75

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 75

cuacaguuua gaagauuua

19

<210> 76

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 76

uaaaucuucu aaacuguagu a

21

<210> 77

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 77

cuacaguuua gaagauuua

19

<210> 78

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 78

uaaaucuucu aaacuguagu a

21

<210> 79

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 79

uacuacaguu uagaagauuu a

21

<210> 80

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 80

uaaaucuucu aaacuguagu aug

23

<210> 81

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 81

uacuacaguu uagaagauuu a

21

<210> 82

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 82

uaaaucuucu aaacuguagu aug

23

<210> 83

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 83

uacuacaguu uagaagauuu a

21

<210> 84

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 84

uaaaucuucu aaacuguagu aug

23

<210> 85

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 85

cuacaguuua gaagauuua

19

<210> 86

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 86

uaaaucuucu aaacuguagu a

21

<210> 87

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 87

cuacaguuua gaagauuua

19

<210> 88

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 88

uaaaucuucu aaacuguagu a

21

<210> 89

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 89

cuacaguuua gaagauuua

19

<210> 90

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 90

uaaaucuucu aaacuguagu a

21

<210> 91

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 91

uacuacaguu uagaagauuu a

21

<210> 92

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 92

uaaaucuucu aaacuguagu aug

23

<210> 93

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 93

uacuacaguu uagaagauuu a

21

<210> 94

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 94

uaaaucuucu aaacuguagu aug

23

<210> 95

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 95

uacuacaguu uagaagauuu a

21

<210> 96

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 96

uaaaucuucu aaacuguagu aug

23

<210> 97

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 97

cuacaguuua gaagauuua

19

<210> 98

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 98

uaaaucuucu aaacuguagu a

21

<210> 99

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 99

cuacaguuua gaagauuua

19

<210> 100

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 100

uaaaucuucu aaacuguagu a

21

<210> 101

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 101

cuacaguuua gaagauuua

19

<210> 102

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 102

uaaaucuucu aaacuguagu a

21

<210> 103

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 103

uacuacaguu uagaagauuu a

21

<210> 104

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 104

uaaaucuucu aaacuguagu aug

23

<210> 105

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 105

uacuacaguu uagaagauuu a

21

<210> 106

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 106

uaaaucuucu aaacuguagu aug

23

<210> 107

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 107

uacuacaguu uagaagauuu a

21

<210> 108

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 108

uaaaucuucu aaacuguagu aug

23

<210> 109

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 109

cuacaguuua gaagauuua

19

<210> 110

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 110

uaaaucuucu aaacuguagu a

21

<210> 111

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 111

cuacaguuua gaagauuua

19

<210> 112

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 112

uaaaucuucu aaacuguagu a

21

<210> 113

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 113

cuacaguuua gaagauuua

19

<210> 114

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 114

uaaaucuucu aaacuguagu a

21

<210> 115

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 115

uacuacaguu uagaagauuu a

21

<210> 116

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 116

uaaaucuucu aaacuguagu aug

23

<210> 117

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 117

uacuacaguu uagaagauuu a

21

<210> 118

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 118

uaaaucuucu aaacuguagu aug

23

<210> 119

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 119

uacuacaguu uagaagauuu a

21

<210> 120

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 120

uaaaucuucu aaacuguagu aug

23

<210> 121

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n represents Z9, and Z9 represents A

<400> 121

ggaagguuac cgagcaaun

19

<210> 122

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z10, and Z10 represents U

<400> 122

nauugcucgg uaaccuucc

19

<210> 123

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is Z11, and Z11 is selected from A, U, G or C

<400> 123

ggaagguuac cgagcaaun

19

<210> 124

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> N represents Z12, Z12 represents a nucleotide,

complementary to Z11, and Z11 is selected from A, U, G or C

<400> 124

nauugcucgg uaaccuucc

19

<210> 125

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is Z11, and Z11 is selected from A, U, G or C

<400> 125

ggaagguuac cgagcaaun

19

<210> 126

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> N represents Z12, Z12 represents a nucleotide,

complementary to Z11, and Z11 is selected from A, U, G or C

<400> 126

nauugcucgg uaaccuuccc u

21

<210> 127

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is Z11, and Z11 is selected from A, U, G or C

<400> 127

agggaagguu acgagcaau n

21

<210> 128

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> N represents Z12, Z12 represents a nucleotide,

complementary to Z11, and Z11 is selected from A, U, G or C

<400> 128

nauugcucgg uaaccuuccc ugg

23

<210> 129

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 129

ggaagguuac cgagcaaua

19

<210> 130

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 130

uauugcucgg uaaccuuccc u

21

<210> 131

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 131

agggaagguu acgagcaau a

21

<210> 132

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 132

uauugcucgg uaaccuuccc ugg

23

<210> 133

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 133

ggaagguuac cgagcaaua

19

<210> 134

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 134

uauugcucgg uaaccuuccc u

21

<210> 135

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 135

ggaagguuac cgagcaaua

19

<210> 136

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 136

uauugcucgg uaaccuuccc u

21

<210> 137

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 137

ggaagguuac cgagcaaua

19

<210> 138

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 138

uauugcucgg uaaccuuccc u

21

<210> 139

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 139

agggaagguu acgagcaau a

21

<210> 140

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 140

uauugcucgg uaaccuuccc ugg

23

<210> 141

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 141

agggaagguu acgagcaau a

21

<210> 142

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 142

uauugcucgg uaaccuuccc ugg

23

<210> 143

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 143

agggaagguu acgagcaau a

21

<210> 144

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 144

uauugcucgg uaaccuuccc ugg

23

<210> 145

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 145

ggaagguuac cgagcaaua

19

<210> 146

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 146

uauugcucgg uaaccuuccc u

21

<210> 147

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 147

ggaagguuac cgagcaaua

19

<210> 148

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 148

uauugcucgg uaaccuuccc u

21

<210> 149

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 149

ggaagguuac cgagcaaua

19

<210> 150

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 150

uauugcucgg uaaccuuccc u

21

<210> 151

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 151

agggaagguu acgagcaau a

21

<210> 152

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 152

uauugcucgg uaaccuuccc ugg

23

<210> 153

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 153

agggaagguu acgagcaau a

21

<210> 154

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 154

uauugcucgg uaaccuuccc ugg

23

<210> 155

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 155

agggaagguu acgagcaau a

21

<210> 156

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 156

uauugcucgg uaaccuuccc ugg

23

<210> 157

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 157

ggaagguuac cgagcaaua

19

<210> 158

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 158

uauugcucgg uaaccuuccc u

21

<210> 159

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 159

ggaagguuac cgagcaaua

19

<210> 160

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 160

uauugcucgg uaaccuuccc u

21

<210> 161

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 161

ggaagguuac cgagcaaua

19

<210> 162

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 162

uauugcucgg uaaccuuccc u

21

<210> 163

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 163

agggaagguu acgagcaau a

21

<210> 164

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 164

uauugcucgg uaaccuuccc ugg

23

<210> 165

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 165

agggaagguu acgagcaau a

21

<210> 166

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 166

uauugcucgg uaaccuuccc ugg

23

<210> 167

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 167

agggaagguu acgagcaau a

21

<210> 168

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 168

uauugcucgg uaaccuuccc ugg

23

<210> 169

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 169

ggaagguuac cgagcaaua

19

<210> 170

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 170

uauugcucgg uaaccuuccc u

21

<210> 171

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 171

ggaagguuac cgagcaaua

19

<210> 172

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 172

uauugcucgg uaaccuuccc u

21

<210> 173

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 173

ggaagguuac cgagcaaua

19

<210> 174

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 174

uauugcucgg uaaccuuccc u

21

<210> 175

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 175

agggaagguu acgagcaau a

21

<210> 176

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 176

uauugcucgg uaaccuuccc ugg

23

<210> 177

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 177

agggaagguu acgagcaau a

21

<210> 178

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 178

uauugcucgg uaaccuuccc ugg

23

<210> 179

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 179

agggaagguu acgagcaau a

21

<210> 180

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 180

uauugcucgg uaaccuuccc ugg

23

<210> 181

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> N is Z13, and Z13 is A

<400> 181

agaacagaca gcagaauun

19

<210> 182

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> N represents Z14, and Z14 represents U

<400> 182

naauucugcu gucuguucu

19

<210> 183

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n represents Z15, and Z15 is selected from A, U, G or C

<400> 183

agaacagaca gcagaauun

19

<210> 184

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> N represents Z16, Z16 represents a nucleotide,

complementary to Z15, and Z15 selected from A, U, G or C

<400> 184

naauucugcu gucuguucu

19

<210> 185

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n represents Z15, and Z15 is selected from A, U, G or C

<400> 185

agaacagaca gcagaauun

19

<210> 186

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> N represents Z16, Z16 represents a nucleotide,

complementary to Z15, and Z15 selected from A, U, G or C

<400> 186

naauucugcu gucuguucuc c

21

<210> 187

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (21)..(21)

<223> n represents Z15, and Z15 is selected from A, U, G or C

<400> 187

ggagaacaga cagcagaauu n

21

<210> 188

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> N represents Z16, Z16 represents a nucleotide,

complementary to Z15, and Z15 selected from A, U, G or C

<400> 188

naauucugcu gucuguucuc cug

23

<210> 189

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 189

agaacagaca gcagaauua

19

<210> 190

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 190

uaauucugcu gucuguucucc

21

<210> 191

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 191

ggagaacaga cagcagaauu a

21

<210> 192

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 192

uaauucugcu gucuguucuc cug

23

<210> 193

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 193

agaacagaca gcagaauua

19

<210> 194

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 194

uaauucugcu gucuguucucc

21

<210> 195

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 195

agaacagaca gcagaauua

19

<210> 196

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 196

uaauucugcu gucuguucucc

21

<210> 197

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 197

agaacagaca gcagaauua

19

<210> 198

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 198

uaauucugcu gucuguucucc

21

<210> 199

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 199

ggagaacaga cagcagaauu a

21

<210> 200

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 200

uaauucugcu gucuguucuc cug

23

<210> 201

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 201

ggagaacaga cagcagaauu a

21

<210> 202

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 202

uaauucugcu gucuguucuc cug

23

<210> 203

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 203

ggagaacaga cagcagaauu a

21

<210> 204

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 204

uaauucugcu gucuguucuc cug

23

<210> 205

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 205

agaacagaca gcagaauua

19

<210> 206

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 206

uaauucugcu gucuguucucc

21

<210> 207

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 207

agaacagaca gcagaauua

19

<210> 208

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 208

uaauucugcu gucuguucucc

21

<210> 209

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 209

agaacagaca gcagaauua

19

<210> 210

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 210

uaauucugcu gucuguucucc

21

<210> 211

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 211

ggagaacaga cagcagaauu a

21

<210> 212

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 212

uaauucugcu gucuguucuc cug

23

<210> 213

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 213

ggagaacaga cagcagaauu a

21

<210> 214

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 214

uaauucugcu gucuguucuc cug

23

<210> 215

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 215

ggagaacaga cagcagaauu a

21

<210> 216

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 216

uaauucugcu gucuguucuc cug

23

<210> 217

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 217

agaacagaca gcagaauua

19

<210> 218

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 218

uaauucugcu gucuguucucc

21

<210> 219

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 219

agaacagaca gcagaauua

19

<210> 220

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 220

uaauucugcu gucuguucucc

21

<210> 221

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 221

agaacagaca gcagaauua

19

<210> 222

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 222

uaauucugcu gucuguucucc

21

<210> 223

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 223

ggagaacaga cagcagaauu a

21

<210> 224

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 224

uaauucugcu gucuguucuc cug

23

<210> 225

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 225

ggagaacaga cagcagaauu a

21

<210> 226

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 226

uaauucugcu gucuguucuc cug

23

<210> 227

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 227

ggagaacaga cagcagaauu a

21

<210> 228

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 228

uaauucugcu gucuguucuc cug

23

<210> 229

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 229

agaacagaca gcagaauua

19

<210> 230

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 230

uaauucugcu gucuguucucc

21

<210> 231

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 231

agaacagaca gcagaauua

19

<210> 232

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 232

uaauucugcu gucuguucucc

21

<210> 233

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 233

agaacagaca gcagaauua

19

<210> 234

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 234

uaauucugcu gucuguucucc

21

<210> 235

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 235

ggagaacaga cagcagaauu a

21

<210> 236

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 236

uaauucugcu gucuguucuc cug

23

<210> 237

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 237

ggagaacaga cagcagaauu a

21

<210> 238

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 238

uaauucugcu gucuguucuc cug

23

<210> 239

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 239

ggagaacaga cagcagaauu a

21

<210> 240

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 240

uaauucugcu gucuguucuc cug

23

<210> 241

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223>n is Z17, and Z17 is A

<400> 241

ccaagaagaa cgcugcaan

19

<210> 242

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z18, and Z18 represents U

<400> 242

nuugcagcgu ucuucuugg

19

<210> 243

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is Z19, and Z19 is selected from A, U, G or C

<400> 243

ccaagaagaa cgcugcaan

19

<210> 244

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z20, Z20 represents a nucleotide,

complementary to Z19, and Z19 selected from A, U, G or C

<400> 244

nuugcagcgu ucuucuugg

19

<210> 245

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n is Z19, and Z19 is selected from A, U, G or C

<400> 245

ccaagaagaa cgcugcaan

19

<210> 246

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z20, Z20 represents a nucleotide,

complementary to Z19, and Z19 selected from A, U, G or C

<400> 246

nuugcagcgu ucuucuuggc c

21

<210> 247

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (21)..(21)

<223> n is Z19, and Z19 is selected from A, U, G or C

<400> 247

ggccaagaag aacgcugcaa n

21

<210> 248

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z20, Z20 represents a nucleotide,

complementary to Z19, and Z19 selected from A, U, G or C

<400> 248

nuugcagcgu ucuucuuggc cug

23

<210> 249

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 249

ccaagaagaa cgcugcaaa

19

<210> 250

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 250

uuugcagcgu ucuucuuggc c

21

<210> 251

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 251

ggccaagaag aacgcugcaa a

21

<210> 252

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 252

uuugcagcgu ucuucuuggc cug

23

<210> 253

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 253

ccaagaagaa cgcugcaaa

19

<210> 254

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 254

uuugcagcgu ucuucuuggc c

21

<210> 255

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 255

ccaagaagaa cgcugcaaa

19

<210> 256

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 256

uuugcagcgu ucuucuuggc c

21

<210> 257

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 257

ccaagaagaa cgcugcaaa

19

<210> 258

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 258

uuugcagcgu ucuucuuggc c

21

<210> 259

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 259

ggccaagaag aacgcugcaa a

21

<210> 260

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 260

uuugcagcgu ucuucuuggc cug

23

<210> 261

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 261

ggccaagaag aacgcugcaa a

21

<210> 262

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 262

uuugcagcgu ucuucuuggc cug

23

<210> 263

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 263

ggccaagaag aacgcugcaa a

21

<210> 264

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 264

uuugcagcgu ucuucuuggc cug

23

<210> 265

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 265

ccaagaagaa cgcugcaaa

19

<210> 266

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 266

uuugcagcgu ucuucuuggc c

21

<210> 267

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 267

ccaagaagaa cgcugcaaa

19

<210> 268

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 268

uuugcagcgu ucuucuuggc c

21

<210> 269

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 269

ccaagaagaa cgcugcaaa

19

<210> 270

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 270

uuugcagcgu ucuucuuggc c

21

<210> 271

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 271

ggccaagaag aacgcugcaa a

21

<210> 272

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 272

uuugcagcgu ucuucuuggc cug

23

<210> 273

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 273

ggccaagaag aacgcugcaa a

21

<210> 274

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 274

uuugcagcgu ucuucuuggc cug

23

<210> 275

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 275

ggccaagaag aacgcugcaa a

21

<210> 276

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 276

uuugcagcgu ucuucuuggc cug

23

<210> 277

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 277

ccaagaagaa cgcugcaaa

19

<210> 278

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 278

uuugcagcgu ucuucuuggc c

21

<210> 279

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 279

ccaagaagaa cgcugcaaa

19

<210> 280

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 280

uuugcagcgu ucuucuuggc c

21

<210> 281

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 281

ccaagaagaa cgcugcaaa

19

<210> 282

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 282

uuugcagcgu ucuucuuggc c

21

<210> 283

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 283

ggccaagaag aacgcugcaa a

21

<210> 284

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 284

uuugcagcgu ucuucuuggc cug

23

<210> 285

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 285

ggccaagaag aacgcugcaa a

21

<210> 286

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 286

uuugcagcgu ucuucuuggc cug

23

<210> 287

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 287

ggccaagaag aacgcugcaa a

21

<210> 288

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 288

uuugcagcgu ucuucuuggc cug

23

<210> 289

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 289

ccaagaagaa cgcugcaaa

19

<210> 290

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 290

uuugcagcgu ucuucuuggc c

21

<210> 291

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 291

ccaagaagaa cgcugcaaa

19

<210> 292

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 292

uuugcagcgu ucuucuuggc c

21

<210> 293

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 293

ccaagaagaa cgcugcaaa

19

<210> 294

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 294

uuugcagcgu ucuucuuggc c

21

<210> 295

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 295

ggccaagaag aacgcugcaa a

21

<210> 296

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 296

uuugcagcgu ucuucuuggc cug

23

<210> 297

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 297

ggccaagaag aacgcugcaa a

21

<210> 298

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 298

uuugcagcgu ucuucuuggc cug

23

<210> 299

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 299

ggccaagaag aacgcugcaa a

21

<210> 300

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 300

uuugcagcgu ucuucuuggc cug

23

<210> 301

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223> n represents Z21, and Z21 represents A

<400> 301

ccaguaagca agccagaan

19

<210> 302

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z22, and Z22 represents U

<400> 302

nuucuggcuu gcuuacugg

19

<210> 303

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223>n is Z23, and Z23 is selected from A, U, G or C

<400> 303

ccaguaagca agccagaan

19

<210> 304

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z24, Z24 represents a nucleotide,

complementary to Z23, and Z23 is selected from A, U, G or C

<400> 304

nuucuggcuu gcuuacugg

19

<210> 305

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (19)..(19)

<223>n is Z23, and Z23 is selected from A, U, G or C

<400> 305

ccaguaagca agccagaan

19

<210> 306

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z24, Z24 represents a nucleotide,

complementary to Z23, and Z23 is selected from A, U, G or C

<400> 306

nuucuggcuu gcuuacuggu a

21

<210> 307

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (21)..(21)

<223>n is Z23, and Z23 is selected from A, U, G or C

<400> 307

uaccaguaag caagccagaa n

21

<210> 308

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Z24, Z24 represents a nucleotide,

complementary to Z23, and Z23 is selected from A, U, G or C

<400> 308

nuucuggcuu gcuuacuggu aac

23

<210> 309

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 309

ccaguaagca agccagaaa

19

<210> 310

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 310

uuucuggcuu gcuuacuggu a

21

<210> 311

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 311

uaccaguaag caagccagaa a

21

<210> 312

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 312

uuucuggcuu gcuuacuggu aac

23

<210> 313

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 313

ccaguaagca agccagaaa

19

<210> 314

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 314

uuucuggcuu gcuuacuggu a

21

<210> 315

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 315

ccaguaagca agccagaaa

19

<210> 316

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 316

uuucuggcuu gcuuacuggu a

21

<210> 317

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 317

ccaguaagca agccagaaa

19

<210> 318

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 318

uuucuggcuu gcuuacuggu a

21

<210> 319

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 319

uaccaguaag caagccagaa a

21

<210> 320

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 320

uuucuggcuu gcuuacuggu aac

23

<210> 321

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 321

uaccaguaag caagccagaa a

21

<210> 322

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 322

uuucuggcuu gcuuacuggu aac

23

<210> 323

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 323

uaccaguaag caagccagaa a

21

<210> 324

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 324

uuucuggcuu gcuuacuggu aac

23

<210> 325

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 325

ccaguaagca agccagaaa

19

<210> 326

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 326

uuucuggcuu gcuuacuggu a

21

<210> 327

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 327

ccaguaagca agccagaaa

19

<210> 328

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 328

uuucuggcuu gcuuacuggu a

21

<210> 329

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 329

ccaguaagca agccagaaa

19

<210> 330

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 330

uuucuggcuu gcuuacuggu a

21

<210> 331

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 331

uaccaguaag caagccagaa a

21

<210> 332

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 332

uuucuggcuu gcuuacuggu aac

23

<210> 333

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 333

uaccaguaag caagccagaa a

21

<210> 334

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 334

uuucuggcuu gcuuacuggu aac

23

<210> 335

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 335

uaccaguaag caagccagaa a

21

<210> 336

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 336

uuucuggcuu gcuuacuggu aac

23

<210> 337

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 337

ccaguaagca agccagaaa

19

<210> 338

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 338

uuucuggcuu gcuuacuggu a

21

<210> 339

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 339

ccaguaagca agccagaaa

19

<210> 340

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 340

uuucuggcuu gcuuacuggu a

21

<210> 341

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 341

ccaguaagca agccagaaa

19

<210> 342

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 342

uuucuggcuu gcuuacuggu a

21

<210> 343

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 343

uaccaguaag caagccagaa a

21

<210> 344

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 344

uuucuggcuu gcuuacuggu aac

23

<210> 345

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 345

uaccaguaag caagccagaa a

21

<210> 346

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 346

uuucuggcuu gcuuacuggu aac

23

<210> 347

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 347

uaccaguaag caagccagaa a

21

<210> 348

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 348

uuucuggcuu gcuuacuggu aac

23

<210> 349

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 349

ccaguaagca agccagaaa

19

<210> 350

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 350

uuucuggcuu gcuuacuggu a

21

<210> 351

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 351

ccaguaagca agccagaaa

19

<210> 352

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 352

uuucuggcuu gcuuacuggu a

21

<210> 353

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 353

ccaguaagca agccagaaa

19

<210> 354

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 354

uuucuggcuu gcuuacuggu a

21

<210> 355

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 355

uaccaguaag caagccagaa a

21

<210> 356

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 356

uuucuggcuu gcuuacuggu aac

23

<210> 357

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 357

uaccaguaag caagccagaa a

21

<210> 358

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 358

uuucuggcuu gcuuacuggu aac

23

<210> 359

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 359

uaccaguaag caagccagaa a

21

<210> 360

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 360

uuucuggcuu gcuuacuggu aac

23

<210> 361

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 361

cuucauucau acagacaaa

19

<210> 362

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 362

uuugucugua ugaaugaaga g

21

<210> 363

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 363

cuacaguuua gaagauuua

19

<210> 364

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 364

uaaaucuucu aaacuguagu a

21

<210> 365

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 365

ggaagguuac cgagcaaua

19

<210> 366

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 366

uauugcucgg uaaccuuccc u

21

<210> 367

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 367

agaacagaca gcagaauua

19

<210> 368

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 368

uaauucugcu gucuguucucc

21

<210> 369

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 369

ccaagaagaa cgcugcaaa

19

<210> 370

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 370

uuugcagcgu ucuucuuggc c

21

<210> 371

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 371

ccaguaagca agccagaaa

19

<210> 372

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 372

uuucuggcuu gcuuacuggu a

21

<210> 373

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 373

cuucauucau acagacaaa

19

<210> 374

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 374

uuugucugua ugaaugaaga g

21

<210> 375

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 375

cuacaguuua gaagauuua

19

<210> 376

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 376

uaaaucuucu aaacuguagu a

21

<210> 377

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 377

ggaagguuac cgagcaaua

19

<210> 378

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 378

uauugcucgg uaaccuuccc u

21

<210> 379

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 379

agaacagaca gcagaauua

19

<210> 380

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 380

uaauucugcu gucuguucucc

21

<210> 381

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 381

ccaagaagaa cgcugcaaa

19

<210> 382

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 382

uuugcagcgu ucuucuuggc c

21

<210> 383

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 383

ccaguaagca agccagaaa

19

<210> 384

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> siRNA

<400> 384

uuucuggcuu gcuuacuggu a

21

<210> 385

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 385

atcaggccag ggaaggttac

20

<210> 386

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 386

tcgggatgaa ggaaccatgt

20

<210> 387

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 387

ggtcggagtc aacggattt

19

<210> 388

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 388

ccagcatcgc cccacttga

19

<---

Claims (601)

1. An siRNA conjugate having the structure represented by Formula (308) where n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4; each of m1, m2 and m3 independently represents an integer from 2 to 10; each of R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ or R ₁₅ is independently selected from H, methyl and ethyl; R ₃ represents a group having the structure represented by Formula A59 where E ₁ represents OH, SH or BH ₂ ; And Nu is an siRNA; the specified siRNA contains a sense strand and an antisense strand, each nucleotide in the specified siRNA independently represents a modified or unmodified nucleotide, where the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II; nucleotide sequence I and nucleotide sequence II are at least partially reverse complementary, forming a double-stranded region; and nucleotide sequence I and nucleotide sequence II are selected from the group of sequences according to the following i) to vi): i) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 1, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 2 and differs from it by no more than three nucleotides: 5'-CUUCAUUCAUCAGACAAZ ₁ -3' (SEQ ID NO: 1); 5'-Z ₂ UUGUCUGUAUGAAUGAAG-3' (SEQ ID NO: 2), where Z ₁ is A, Z ₂ is U, nucleotide sequence I contains nucleotide Z ₃ at the site corresponding to Z ₁ , nucleotide sequence II contains nucleotide Z ₄ at the site corresponding to Z ₂ , and Z ₄ is the first nucleotide with 5 '-end of the antisense strand; ii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 61, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 62 and differs from it by no more than three nucleotides: 5'-CUACAGUUUAGAAGAUUUZ ₅ -3' (SEQ ID NO: 61); 5'-Z ₆ AAAUCUUCUAAACUGUAG-3' (SEQ ID NO: 62), where Z ₅ is A, Z ₆ is U, nucleotide sequence I contains nucleotide Z ₇ at the site corresponding to Z ₅ , nucleotide sequence II contains nucleotide Z ₈ at the site corresponding to Z ₆ , and Z ₈ represents the first nucleotide with 5 '-end of the antisense strand; iii) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 121, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 122 and differs from it by no more than three nucleotides: 5'-GGAAGGUUACCGAGCAAUZ ₉ -3' (SEQ ID NO: 121); 5'-Z ₁₀ AUUGCUCGGUAACCUUCC-3' (SEQ ID NO: 122), where Z ₉ is A, Z ₁₀ is U, nucleotide sequence I contains nucleotide Z ₁₁ at the site corresponding to Z ₉ , nucleotide sequence II contains nucleotide Z ₁₂ at the site corresponding to Z ₁₀ , and Z ₁₂ represents the first nucleotide with 5 '-end of the antisense strand; iv) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 181, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 182 and differs from it by no more than three nucleotides: 5'-AGAACAGACAGCAGAUUZ ₁₃ -3' (SEQ ID NO: 181); 5'-Z ₁₄ AAUUCUGCUGUCUGUUCU-3' (SEQ ID NO: 182), where Z ₁₃ is A, Z ₁₄ is U, nucleotide sequence I contains nucleotide Z ₁₅ at the site corresponding to Z ₁₃ , nucleotide sequence II contains nucleotide Z ₁₆ at the site corresponding to Z ₁₄ , and Z ₁₆ represents the first nucleotide with 5 '-end of the antisense strand; v) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 241, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 242 and differs from it by no more than three nucleotides: 5'-CCAAGAAGAACGCUGCAAZ ₁₇ -3' (SEQ ID NO: 241); 5'-Z ₁₈ UUGCAGCGUUCUUCUUGG-3' (SEQ ID NO: 242), where Z ₁₇ is A, Z ₁₈ is U, nucleotide sequence I contains nucleotide Z ₁₉ at the site corresponding to Z ₁₇ , nucleotide sequence II contains nucleotide Z ₂₀ at the site corresponding to Z ₁₈ , and Z ₂₀ represents the first nucleotide with 5 '-end of the antisense strand; And vi) nucleotide sequence I has the same length as the nucleotide sequence presented in SEQ ID NO: 301, and differs from it by no more than three nucleotides; and nucleotide sequence II has the same length as the nucleotide sequence set forth in SEQ ID NO: 302 and differs from it by no more than three nucleotides: 5'-CCAGUAAGCAAGCCAGAAZ ₂₁ -3' (SEQ ID NO: 301); 5'-Z ₂₂ UUCUGGCUUGCUUACUGG-3' (SEQ ID NO: 302), where Z ₂₁ is A, Z ₂₂ is U, nucleotide sequence I contains nucleotide Z ₂₃ at the site corresponding to Z ₂₁ , nucleotide sequence II contains nucleotide Z ₂₄ at the site corresponding to Z ₂₂ , and Z ₂₄ is the first nucleotide with 5 '-end of the antisense strand; R ₂ is a linear alkylene of 1 to 20 carbon atoms in length, where one or more carbon atoms are optionally substituted by any one or more of the group consisting of: C(O), NH, O, S, CH=N, S(O ) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene, and where R ₂ is optionally substituted by any one or more of the group , consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl-NH ₂ , -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -NH(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkylphenyl), -NH(C ₁ -C ₁₀ alkylphenyl), cyano, nitro, -CO ₂ H, -C(O)O( C ₁ -C ₁₀ alkyl), -CON(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ alkyl)C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)C( O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ -C ₁₀ haloalkyl, -OC(O)C ₁ - C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH(phenyl), -NHSO ₂ (C ₁ -C ₁₀ alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C ₁ -C ₁₀ haloalkyl); each L ₁ is independently a linear alkylene of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally substituted by any one or more of the group consisting of: C(O), NH, O, S, CH=N, S (O) ₂ , C ₂ -C ₁₀ alkenylene, C ₂ -C ₁₀ alkynylene, C ₆ -C ₁₀ arylene, C ₃ -C ₁₈ heterocyclylene and C ₅ -C ₁₀ heteroarylene; and wherein L ₁ is optionally substituted by any one or more of the group consisting of: C ₁ -C ₁₀ alkyl, C ₆ -C ₁₀ aryl, C ₅ -C ₁₀ heteroaryl, C ₁ -C ₁₀ haloalkyl, -OC ₁ -C ₁₀ alkyl, -OC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ haloalkyl, -SC ₁ -C ₁₀ alkyl, -SC ₁ -C ₁₀ alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ haloalkyl, halogen substituent, -OH, -SH, -NH ₂ , -C ₁ -C ₁₀ alkyl-NH ₂ , -N(C ₁ -C ₁₀ alkyl)(C ₁ - C ₁₀ alkyl), -NH(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkylphenyl), -NH(C ₁ -C ₁₀ alkylphenyl), cyano, nitro, -CO ₂ H, -C(O)O(C ₁ -C ₁₀ alkyl), -CON(C ₁ -C ₁₀ alkyl)(C ₁ -C ₁₀ alkyl), -CONH(C ₁ -C ₁₀ alkyl), -CONH ₂ , -NHC(O)(C ₁ -C ₁₀ alkyl), -NHC(O)(phenyl), -N(C ₁ -C ₁₀ alkyl)C(O)(C ₁ -C ₁₀ alkyl), -N(C ₁ -C ₁₀ alkyl)C(O)(phenyl), -C(O)C ₁ -C ₁₀ alkyl, -C(O)C ₁ -C ₁₀ alkylphenyl, -C(O)C ₁ - C ₁₀ haloalkyl, -OC(O)C ₁ -C ₁₀ alkyl, -SO ₂ (C ₁ -C ₁₀ alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ haloalkyl), -SO ₂ NH ₂ , -SO ₂ NH (C ₁ -C ₁₀ alkyl), -SO ₂ NH (phenyl), -NHSO ₂ (C ₁ -C ₁₀ alkyl), -NHSO ₂ (phenyl) and -NHSO ₂ (C ₁ - C ₁₀ haloalkyl), and L ₁ is a combination of compounds of at least two groups A1, A4, A8, A10 and A11: where each j1 independently represents an integer from 1 to 20; and each j2 is independently an integer from 1 to 20; And denotes the site of covalent attachment of a group; And M ₁ is a targeting group, wherein each targeting group is independently a ligand that binds to an asialoglycoprotein receptor on the surface of a mammalian hepatocyte. 2. The siRNA conjugate according to claim 1, where the length L ₁ is 3-25 atoms; or the length L ₁ is 4-15 atoms. 3. The siRNA conjugate according to claim 1 or 2, where each of m1, m2 and m3 independently represents an integer from 2 to 5 and/or m1=m2=m3. 4. The siRNA conjugate according to any one of paragraphs. 1-3, wherein each targeting group is independently an asialoglycoprotein or a saccharide; or each targeting group is independently selected from one of the following: D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D -mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose , α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β -D-glucofuranose, methyl-2,3,4-tris-O-acetyl-1-thio-6-O-trityl-α-D-glucofuranose, 4-thio-β-D-galactopyranose, ethyl-3,4 ,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L -ribose and L-4-thioribose; or at least one or each targeting group is galactose or N-acetylgalactosamine. 5. The siRNA conjugate according to any one of paragraphs. 1-4, where R ₂ contains both a binding site for the N atom on the nitrogen-containing backbone and a binding site for the P atom in R ₃ ; or in R ₂ the binding site to the N atom on the nitrogen-containing backbone forms an amide bond to the N atom, and the binding site to the P atom in R ₃ forms a phosphoester bond to the P atom; or R ₂ is selected from the groups represented by Formulas (B5), (B6), (B5') or (B6'): Where denotes the site of covalent attachment of a group; And q2 represents an integer from 1 to 10. 6. The siRNA conjugate according to any one of paragraphs. 1-5, having the structure represented by Formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), ( 413), (414), (415), (416), (417), (418), (419), (420), (421) or (422): 7. The siRNA conjugate according to any one of paragraphs. 1-6, wherein the P atom in Formula A59 is associated with a terminal region of the sense strand or antisense strand of the siRNA, and the terminal region refers to the first four nucleotides closest to either end of the sense strand or antisense strand; or the P atom in Formula A59 is associated with the end of the sense strand or antisense strand of the siRNA; or the P atom in Formula A59 is associated with the 3' end of the sense strand of the siRNA; or the P atom in Formula A59 is linked to the 2', 3' or 5' nucleotide position in the siRNA by a phosphodiester bond. 8. The siRNA conjugate according to claim 1, where i) the nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 1 by no more than one nucleotide; and/or the nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 2 by no more than one nucleotide; or ii) nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 61 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 62 by no more than one nucleotide ; or iii) nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 121 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 122 by no more than one nucleotide ; or iv) nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 181 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 182 by no more than one nucleotide ; or v) nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 241 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 242 by no more than one nucleotide ; or vi) nucleotide sequence I differs from the nucleotide sequence presented in SEQ ID NO: 301 by no more than one nucleotide, and/or nucleotide sequence II differs from the nucleotide sequence presented in SEQ ID NO: 302 by no more than one nucleotide . 9. The siRNA conjugate according to claim 1 or 8, where i) the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 2 includes a difference in the Z ₄ site, and Z ₄ is selected from A, C or G; or ii) the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 62 includes a difference in the Z ₈ site, and Z ₈ is selected from A, C or G; or iii) the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 122 includes a difference in the Z ₁₂ site, and Z ₁₂ is selected from A, C or G; or iv) the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 182 includes a difference in the Z ₁₆ site, and Z ₁₆ is selected from A, C or G; or v) the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 242 includes a difference in the Z ₂₀ site, and Z ₂₀ is selected from A, C or G; or vi) the nucleotide difference between nucleotide sequence II and the nucleotide sequence presented in SEQ ID NO: 302 includes a difference in the Z ₂₄ site, and Z ₂₄ is selected from A, C or G, where Z ₃ represents a nucleotide complementary to Z ₄ ; or Z ₇ is a nucleotide complementary to Z ₈ ; or Z ₁₁ is a nucleotide complementary to Z ₁₂ ; or Z ₁₅ is a nucleotide complementary to Z ₁₆ ; or Z ₁₉ is a nucleotide complementary to Z ₂₀ ; or Z ₂₃ is a nucleotide complementary to Z ₂₄ . 10. The siRNA conjugate according to claim 8 or 9, where the sense strand additionally contains nucleotide sequence III, the antisense strand additionally contains nucleotide sequence IV, each of nucleotide sequence III and nucleotide sequence IV independently has a length of 1-4 nucleotides, nucleotide sequence III is associated with At the 5' end of nucleotide sequence I, nucleotide sequence IV is linked to the 3' end of nucleotide sequence II, and nucleotide sequence III is of the same length and is substantially reverse complementary or completely reverse complementary to nucleotide sequence IV; substantially reverse complementary refers to no more than one mispaired base between two nucleotide sequences; and fully reverse complementary refers to the absence of mispairing between two nucleotide sequences. 11. The siRNA conjugate according to claim 10, where i) nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 3, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 4; and nucleotide sequences III and IV are both one nucleotide in length, and the base of nucleotide sequence III is U; or nucleotide sequences III and IV are both two nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CU; or nucleotide sequences III and IV are both three nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UCU; or nucleotide sequences III and IV are both four nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UUCU; or ii) nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 63, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 64; and nucleotide sequences III and IV are both one nucleotide in length and the base of nucleotide sequence III is A; or nucleotide sequences III and IV are both two nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UA; or nucleotide sequences III and IV are both three nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is AUA; or nucleotide sequences III and IV are both four nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CAUA; or iii) nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 123, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 124; and nucleotide sequences III and IV are both one nucleotide in length and the base of nucleotide sequence III is G; or nucleotide sequences III and IV are both two nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is AG; or nucleotide sequences III and IV are both three nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CAG; or nucleotide sequences III and IV are both four nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is CCAG; or iv) nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 183, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 184; and nucleotide sequences III and IV are both one nucleotide in length and the base of nucleotide sequence III is G; or nucleotide sequences III and IV are both two nucleotides in length and in the 5' to 3' direction the base composition of nucleotide sequence III is GG; or nucleotide sequences III and IV are both three nucleotides in length and in the 5'-end to 3'-end direction the base composition of nucleotide sequence III is AGG; or nucleotide sequences III and IV are both four nucleotides in length and in the 5'-end to 3'-end direction the base composition of nucleotide sequence III is CAGG; or v) nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 243, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 244; and nucleotide sequences III and IV are both one nucleotide in length and the base of nucleotide sequence III is G; or nucleotide sequences III and IV are both two nucleotides in length and in the 5' to 3' direction the base composition of nucleotide sequence III is GG; or nucleotide sequences III and IV are both three nucleotides in length and in the 5'-end to 3'-end direction the base composition of nucleotide sequence III is AGG; or nucleotide sequences III and IV are both four nucleotides in length and in the 5'-end to 3'-end direction the base composition of nucleotide sequence III is CAGG; or vi) nucleotide sequence I is the nucleotide sequence presented in SEQ ID NO: 303, and nucleotide sequence II is the nucleotide sequence presented in SEQ ID NO: 304; and nucleotide sequences III and IV are both one nucleotide in length and the base of nucleotide sequence III is A; or nucleotide sequences III and IV are both two nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UA; or nucleotide sequences III and IV are both three nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is UUA; or nucleotide sequences III and IV are both four nucleotides in length and in the 5'-end to 3'-end direction, the base composition of nucleotide sequence III is GUUA. 12. The siRNA conjugate according to any one of paragraphs. 1-11, wherein the antisense strand further comprises a nucleotide sequence V, wherein the nucleotide sequence V is 1-3 nucleotides in length and linked to the 3' end of the antisense strand to form a 3' overhanging end of the antisense strand; or the V nucleotide sequence is 2 nucleotides long; or the nucleotide sequence V is two thymidine deoxyribonucleotides in a row or two uridine ribonucleotides in a row; or the nucleotide sequence of V is complementary to the nucleotides at the corresponding site of the target mRNA. 13. The siRNA conjugate according to any one of paragraphs. 1-12, where the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 5, and the antisense strand contains the nucleotide sequence presented in SEQ ID NO: 6; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 7, and the antisense strand contains the nucleotide sequence presented in SEQ ID NO: 8; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 65, and the antisense strand contains the nucleotide sequence presented in SEQ ID NO: 66; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 67, and the antisense strand contains the nucleotide sequence presented in SEQ ID NO: 68; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 125, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 126; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 127, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 128; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 185, and the antisense strand contains the nucleotide sequence presented in SEQ ID NO: 186; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 187, and the antisense strand contains the nucleotide sequence presented in SEQ ID NO: 188; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 245, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 246; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 247, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 248; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 305, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 306; or the sense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 307, and the antisense strand of the siRNA contains the nucleotide sequence presented in SEQ ID NO: 308. 14. The siRNA conjugate according to any one of paragraphs. 8-13, where the siRNA has the nucleotide sequence represented in siC5a1, siC5a2, siC5b1, siC5b2, siC5c1, siC5c2, siC5d1, siC5d2, siC5e1, siC5e2, siC5f1 or siC5f2: siC5a1: 5'-CUUCAUUCAUACAGACAAA-3' (SEQ ID NO: 9) 5'-UUUGUCUGUAUGAAUGAAGAG-3' (SEQ ID NO: 10) siC5a2: 5'-CUCUUCAUUCAUACAGACAAA-3' (SEQ ID NO: 11) 5'-UUUGUCUGUAUGAAUGAAGAGAA-3' (SEQ ID NO: 12) siC5b1: 5'-CUACAGUUUAGAAGAUUUA-3' (SEQ ID NO: 69) 5'-UAAAUCUUCUAAACUGUAGUA-3' (SEQ ID NO: 70) siC5b2: 5'-UACUACAGUUAGAAGAUUUA-3' (SEQ ID NO: 71) 5'-UAAAUCUUCUAAACUGUAGUAUG-3' (SEQ ID NO: 72) siC5c1: 5'-GGAAGGUUACCGAGCAAUA-3' (SEQ ID NO: 129) 5'-UAUUGCUCGGUAACCUUCCCU-3' (SEQ ID NO: 130) siC5c2: 5'-AGGGAAGGUUACCGAGCAAUA-3' (SEQ ID NO: 131) 5'-UAUUGCUCGGUAACCUUCCCUGG-3' (SEQ ID NO: 132) siC5d1: 5'-AGAACAGACAGCAGAAUUA-3' (SEQ ID NO: 189) 5'-UAAUUCUGCUGUCUGUUCUCC-3' (SEQ ID NO: 190) siC5d2: 5'-GGAGAACAGACAGCAGAAUUA-3' (SEQ ID NO: 191) 5'-UAAUUCUGCUGUCUGUUCUCCUG-3' (SEQ ID NO: 192) siC5e1: 5'-CCAAGAAGAACGCUGCAAA-3' (SEQ ID NO: 249) 5'-UUUGCAGCGUUCUUCUUGGCC-3' (SEQ ID NO: 250) siC5e2: 5'-GGCCAAGAAGAACGCUGCAAA-3' (SEQ ID NO: 251) 5'-UUUGCAGCGUUCUUCUUGGCCUG-3' (SEQ ID NO: 252) siC5f1: 5'-CCAGUAAGCAAGCCAGAAA-3' (SEQ ID NO: 309) 5'-UUUCUGGCUUGCUUACUGGUA-3' (SEQ ID NO: 310) siC5f2: 5'-UACCAGUAAGCAAGCCAGAAA-3' (SEQ ID NO: 311) 5'-UUUCUGGCUUGCUUACUGGUAAC-3' (SEQ ID NO: 312). 15. The siRNA conjugate according to claim 1, where each nucleotide in the sense strand and the antisense strand is independently a fluorine-modified nucleotide or a nucleotide with a modification other than fluorine; or fluorine-modified nucleotides are located in nucleotide sequence I and nucleotide sequence II; and in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of nucleotide sequence I are fluorine modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of nucleotide sequence II are fluorine modified nucleotides; or in a direction from the 5' end to the 3' end, nucleotides at positions 7, 8 and 9 or 5, 7, 8 and 9 of nucleotide sequence I in the sense strand are fluorine modified nucleotides, and nucleotides at the remaining positions in the sense strand are are nucleotides with a modification other than fluorine; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 or 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand are fluorine modified nucleotides, and the nucleotides at the remaining positions in the antisense chain are nucleotides with a modification other than fluorine. 16. The siRNA conjugate of claim 15, wherein each nucleotide with a non-fluorine modification is independently selected from a nucleotide formed by replacing the 2'-hydroxy group of the ribose group of the nucleotide with a non-fluorine group or an analogue of the nucleotide; or a nucleotide formed by replacing the 2'-hydroxy group of the ribose group of a nucleotide with a group other than fluorine, selected from one of 2'-alkoxy-modified nucleotide, 2'-substituted alkoxy-modified nucleotide, 2'-alkyl-modified nucleotide, 2' -substituted alkyl-modified nucleotide, 2'-amino-modified nucleotide, 2'-substituted amino-modified nucleotide and 2'-deoxynucleotide; and the nucleotide analogue is selected from one of isonucleotide, LNA, ENA, cET, UNA and GNA; or each nucleotide with a modification other than fluorine is a methoxy-modified nucleotide, and methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxy group of a ribose group with a methoxy group. 17. The siRNA conjugate according to claim 15 or 16, where in the direction from the 5'-end to the 3'-end, the nucleotides at positions 5, 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides in the remaining positions in the sense strand of siRNA are methoxy-modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14 and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are are methoxy-modified nucleotides; or in a direction from the 5' end to the 3' end, nucleotides at positions 5, 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides; or in a direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand of the siRNA are methoxy-modified nucleotides. 18. The siRNA conjugate according to any one of paragraphs. 1-17, where the siRNA is any of 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 siC5f2-M3: siC5a1-M1: 5'-CmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 13) 5'-UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm-3' (SEQ ID NO: 14) siC5a2-M1: 5'-CmUmCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 19) 5'-UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm-3' (SEQ ID NO: 20) siC5a1-M2: 5'-CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 27) 5'-UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmsAmsGm-3' (SEQ ID NO: 28) siC5a2-M2: 5'-CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 21) 5'-UmUfUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmAmAm-3' (SEQ ID NO: 22) siC5a1-M3: 5'-CmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 17) 5'-UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm-3' (SEQ ID NO: 18) siC5a2-M3: 5'-CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 23) 5'-UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm-3' (SEQ ID NO: 24) siC5b1-M1: 5'-CmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 73) 5'-UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm-3' (SEQ ID NO: 74) siC5b2-M1: 5'-UmAmCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 79) 5'-UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm-3' (SEQ ID NO: 80) siC5b1-M2: 5'-CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 75) 5'-UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAm-3' (SEQ ID NO: 76) siC5b2-M2: 5'-UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 81) 5'-UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmUmGm-3' (SEQ ID NO: 82) siC5b1-M3: 5'-CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 77) 5'-UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm-3' (SEQ ID NO: 78) siC5b2-M3: 5'-UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 83) 5'-UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm-3' (SEQ ID NO: 84) siC5c1-M1: 5'-GmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 133) 5'-UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm-3' (SEQ ID NO: 134) siC5c2-M1: 5'-AmGmGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 139) 5'-UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm-3' (SEQ ID NO: 140) siC5c1-M2: 5'-GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 135) 5'-UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUm-3' (SEQ ID NO: 136) siC5c2-M2: 5'-AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 141) 5'-UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmGmGm-3' (SEQ ID NO: 142) siC5c1-M3: 5'-GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 137) 5'-UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm-3' (SEQ ID NO: 138) siC5c2-M3: 5'-AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 143) 5'-UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm-3' (SEQ ID NO: 144) siC5d1-M1: 5'-AmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 193) 5'-UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm-3' (SEQ ID NO: 194) siC5d2-M1: 5'-GmGmAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 199) 5'-UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm-3' (SEQ ID NO: 200) siC5d1-M2: 5'-AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 195) 5'-UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCm-3' (SEQ ID NO: 196) siC5d2-M2: 5'-GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 201) 5'-UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmUmGm-3' (SEQ ID NO: 202) siC5d1-M3: 5'-AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 197) 5'-UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm-3' (SEQ ID NO: 198) siC5d2-M3: 5'-GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 203) 5'-UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm-3' (SEQ ID NO: 204) siC5e1-M1: 5'-CmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 253) 5'-UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm-3' (SEQ ID NO: 254) siC5e2-M1: 5'-GmGmCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 259) 5'-UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm-3' (SEQ ID NO: 260) siC5e1-M2: 5'-CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 255) 5'-UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCm-3' (SEQ ID NO: 256) siC5e2-M2: 5'-GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 261) 5'-UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmUmGm-3' (SEQ ID NO: 262) siC5e1-M3: 5'-CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 257) 5'-UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm-3' (SEQ ID NO: 258) siC5e2-M3: 5'-GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 263) 5'-UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm-3' (SEQ ID NO: 264) siC5f1-M1: 5'-CmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 313) 5'-UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm-3' (SEQ ID NO: 314) siC5f2-M1: 5'-UmAmCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 319) 5'-UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm-3' (SEQ ID NO: 320) siC5f1-M2: 5'-CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 315) 5'-UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAm-3' (SEQ ID NO: 316) siC5f2-M2: 5'-UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 321) 5'-UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmAmCm-3' (SEQ ID NO: 322) siC5f1-M3: 5'-CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 317) 5'-UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm-3' (SEQ ID NO: 318) siC5f2-M3: 5'-UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 323) 5'-UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm-3' (SEQ ID NO: 324). 19. The siRNA conjugate according to claim 1, wherein the siRNA at least one phosphate group is a thiophosphate group and said thiophosphate bond is present in at least one of the following positions: the position between the first nucleotide and the second nucleotide at the 5' end of the sense strand; the position between the second nucleotide and the third nucleotide at the 5' end of the sense strand; the position between the first nucleotide and the second nucleotide at the 3' end of the sense strand; the position between the second nucleotide and the third nucleotide at the 3' end of the sense strand; the position between the first nucleotide and the second nucleotide at the 5' end of the antisense strand; the position between the second nucleotide and the third nucleotide at the 5' end of the antisense strand; the position between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; And the position between the second nucleotide and the third nucleotide at the 3' end of the antisense strand. 20. The siRNA conjugate according to any one of paragraphs. 1-19, where the siRNA is any of siC5a1-M1S, siC5a2-M1S, siC5a1-M2S, siC5a2-M2S, siC5a1-M3S, siC5a2-M3S, siC5b1-M1S, siC5b2-M1S, siC5b1-M2S, siC5b2-M2S, siC5b1-M3S, siC5b2-M3S, siC5c1-M1S, siC5c2-M1S, siC5c1-M2S, siC5c2-M2S, siC5c1-M3S, siC5c2-M3S, siC5d1-M1S, siC5d2-M1S, siC5d1-M2S, siC5d2-M2S, siC5d 1- M3S, SIC5D2-M3S, SIC5E1-M1S, SIC5E2-M1S, SIC5E1-M2S, SIC5E2-M2S, SIC5E1-M3S, SIC5E2-M3S, SIC5F1-M1S, SIC5F2-M1S, SIC5F2-M2S, SIC5F1-M3S or SIC iC5f2-M3S: siC5a1-M1S: 5'-CmsUmsUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 25) 5'-UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm-3' (SEQ ID NO: 26) siC5a2-M1S: 5'-CmsUmsCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 31) 5'-UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm-3' (SEQ ID NO: 32) siC5a1-M2S: 5'-CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 27) 5'-UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmsAmsGm-3' (SEQ ID NO: 28) siC5a2-M2S: 5'-CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 33) 5'-UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm-3' (SEQ ID NO: 34) siC5a1-M3S: 5'-CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 29) 5'-UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm-3' (SEQ ID NO: 30) siC5a2-M3S: 5'-CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 35) 5'-UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm-3' (SEQ ID NO: 36) siC5b1-M1S: 5'-CmsUmsAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 85) 5'-UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm-3' (SEQ ID NO: 86) siC5b2-M1S: 5'-UmsAmsCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 91) 5'-UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm-3' (SEQ ID NO: 92) siC5b1-M2S: 5'-CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 87) 5'-UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmsUmsAm-3' (SEQ ID NO: 88) siC5b2-M2S: 5'-UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 93) 5'-UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm-3' (SEQ ID NO: 94) siC5b1-M3S: 5'-CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 89) 5'-UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm-3' (SEQ ID NO: 90) siC5b2-M3S: 5'-UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 95) 5'-UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm-3' (SEQ ID NO: 96) siC5c1-M1S: 5'-GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 145) 5'-UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm-3' (SEQ ID NO: 146) siC5c2-M1S: 5'-AmsGmsGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 151) 5'-UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm-3' (SEQ ID NO: 152) siC5c1-M2S: 5'-GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 147) 5'-UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmsCmsUm-3' (SEQ ID NO: 148) siC5c2-M2S: 5'-AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 153) 5'-UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm-3' (SEQ ID NO: 154) siC5c1-M3S: 5'-GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 149) 5'-UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm-3' (SEQ ID NO: 150) siC5c2-M3S: 5'-AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 155) 5'-UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm-3' (SEQ ID NO: 156) siC5d1-M1S: 5'-AmsGmsAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 205) 5'-UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm-3' (SEQ ID NO: 206) siC5d2-M1S: 5'-GmsGmsAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 211) 5'-UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm-3' (SEQ ID NO: 212) siC5d1-M2S: 5'-AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 207) 5'-UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmsCmsCm-3' (SEQ ID NO: 208) siC5d2-M2S: 5'-GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 213) 5'-UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm-3' (SEQ ID NO: 214) siC5d1-M3S: 5'-AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 209) 5'-UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm-3' (SEQ ID NO: 210) siC5d2-M3S: 5'-GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 215) 5'-UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm-3' (SEQ ID NO: 216) siC5e1-M1S: 5'-CmsCmsAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 265) 5'-UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm-3' (SEQ ID NO: 266) siC5e2-M1S: 5'-GmsGmsCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 271) 5'-UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm-3' (SEQ ID NO: 272) siC5e1-M2S: 5'-CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 267) 5'-UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmsCmsCm-3' (SEQ ID NO: 268) siC5e2-M2S: 5'-GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 273) 5'-UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm-3' (SEQ ID NO: 274) siC5e1-M3S: 5'-CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 269) 5'-UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm-3' (SEQ ID NO: 270) siC5e2-M3S: 5'-GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 275) 5'-UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm-3' (SEQ ID NO: 276) siC5f1-M1S: 5'-CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 325) 5'-UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm-3' (SEQ ID NO: 326) siC5f2-M1S: 5'-UmsAmsCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 331) 5'-UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm-3' (SEQ ID NO: 332) siC5f1-M2S: 5'-CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 327) 5'-UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmsUmsAm-3' (SEQ ID NO: 328) siC5f2-M2S: 5'-UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 333) 5'-UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm-3' (SEQ ID NO: 334) siC5f1-M3S: 5'-CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 329) 5'-UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm-3' (SEQ ID NO: 330) siC5f2-M3S: 5'-UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 335) 5'-UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm-3' (SEQ ID NO: 336). 21. The siRNA conjugate according to any one of paragraphs. 1-20, where the 5'-terminal nucleotide in the antisense strand of the siRNA is a nucleotide with a 5'-phosphate group or a nucleotide modified with an analogue of the 5'-phosphate group; or a nucleotide with a 5'-phosphate group is the nucleotide represented by Formula (2); and the nucleotide modified with a 5'-phosphate group analogue is selected from the nucleotide represented by any of Formulas (3)-(6): where R is selected from H, OH, methoxy or F; and The base is a base selected from A, U, C, G or T. 22. The siRNA conjugate according to any one of paragraphs. 1-21, where Mirnka is any of the SIC5A1-M1P1, SIC5A1-M2P1, SIC5A1-M3P1, SIC5A2-M1P1, SIC5A2-M2P1, SIC5A2-M3P1, SIC5A1-M1SP1, SIC5A1-M2SP1, SIC5A1-M3SP1, SIC5A2 -M1SP1, SIC5A2 -M1S. P1, siC5a2-M2SP1, siC5a2-M3SP1, siC5b1-M1P1, siC5b1-M2P1, siC5b1-M3P1, siC5b2-M1P1, siC5b2-M2P1, siC5b2-M3P1, siC5b1-M1SP1, siC5b1-M2SP1, siC5b1-M3SP1, siC5 b2-M1SP1, siC5b2- M2SP1, siC5b2-M3SP1, siC5c1-M1P1, siC5c1-M2P1, siC5c1-M3P1, siC5c2-M1P1, siC5c2-M2P1, siC5c2-M3P1, siC5c1-M1SP1, siC5c1-M2SP1, siC5c1-M3SP1, siC5c2-M1SP 1, siC5c2-M2SP1, siC5c2-M3SP1, siC5d1-M1P1, siC5d1-M2P1, siC5d1-M3P1, siC5d2-M1P1, siC5d2-M2P1, siC5d2-M3P1, siC5d1-M1SP1, siC5d1-M2SP1, siC5d1-M3SP1, siC5d2-M1SP1, siC5 d2-M2SP1, siC5d2- M3SP1, siC5e1-M1P1, siC5e1-M2P1, siC5e1-M3P1, siC5e2-M1P1, siC5e2-M2P1, siC5e2-M3P1, siC5e1-M1SP1, siC5e1-M2SP1, siC5e1-M3SP1, siC5e2-M1SP1, siC5e2-M2SP 1, siC5e2-M3SP1, siC5f1-M1P1, siC5f1-M2P1, siC5f1-M3P1, siC5f2-M1P1, siC5f2-M2P1, siC5f2-M3P1, siC5f1-M1SP1, siC5f1-M2SP1, siC5f1-M3SP1, siC5f2-M1SP1, siC5f2-M2SP1 or siC5 f2-M3SP1: siC5a1-M1P1: 5'-CmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 37) 5'-P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm-3' (SEQ ID NO: 38) siC5a1-M2P1: 5'-CmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 39) 5'-P1UmUfUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGm-3' (SEQ ID NO: 40) siC5a1-M3P1: 5'-CmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 41) 5'-P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGm-3' (SEQ ID NO: 42) siC5a2-M1P1: 5'-CmUmCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 43) 5'-P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm-3' (SEQ ID NO: 44) siC5a2-M2P1: 5'-CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 45) 5'-P1UmUfUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmAmAm-3' (SEQ ID NO: 46) siC5a2-M3P1: 5'-CmUmCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 47) 5'-P1UmUfUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmAmAm-3' (SEQ ID NO: 48) siC5a1-M1SP1: 5'-CmsUmsUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 49) 5'-P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm-3' (SEQ ID NO: 50) siC5a1-M2SP1: 5'-CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 51) 5'-P1UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmsAmsGm-3' (SEQ ID NO: 52) siC5a1-M3SP1: 5'-CmsUmsUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 53) 5'-P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmsAmsGm-3' (SEQ ID NO: 54) siC5a2-M1SP1: 5'-CmsUmsCmUmUmCmAmUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 55) 5'-P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm-3' (SEQ ID NO: 56) siC5a2-M2SP1: 5'-CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 57) 5'-P1UmsUfsUmGmUmCfUmGfUfAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm-3' (SEQ ID NO: 58) siC5a2-M3SP1: 5'-CmsUmsCmUmUmCmAfUmUfCfAfUmAmCmAmGmAmCmAmAmAm-3' (SEQ ID NO: 59) 5'-P1UmsUfsUmGmUmCfUmGmUmAmUmGmAmAfUmGfAmAmGmAmGmsAmsAm-3' (SEQ ID NO: 60) siC5b1-M1P1: 5'-CmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 97) 5'-P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm-3' (SEQ ID NO: 98) siC5b1-M2P1: 5'-CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 99) 5'-P1UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAm-3' (SEQ ID NO: 100) siC5b1-M3P1: 5'-CmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 101) 5'-P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAm-3' (SEQ ID NO: 102) siC5b2-M1P1: 5'-UmAmCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 103) 5'-P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm-3' (SEQ ID NO: 104) siC5b2-M2P1: 5'-UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 105) 5'-P1UmAfAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmUmGm-3' (SEQ ID NO: 106) siC5b2-M3P1: 5'-UmAmCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 107) 5'-P1UmAfAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmUmGm-3' (SEQ ID NO: 108) siC5b1-M1SP1: 5'-CmsUmsAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 109) 5'-P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm-3' (SEQ ID NO: 110) siC5b1-M2SP1: 5'-CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 111) 5'-P1UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmsUmsAm-3' (SEQ ID NO: 112) siC5b1-M3SP1: 5'-CmsUmsAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 113) 5'-P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmsUmsAm-3' (SEQ ID NO: 114) siC5b2-M1SP1: 5'-UmsAmsCmUmAmCmAmGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 115) 5'-P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm-3' (SEQ ID NO: 116) siC5b2-M2SP1: 5'-UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 117) 5'-P1UmsAfsAmAmUmCfUmUfCfUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm-3' (SEQ ID NO: 118) siC5b2-M3SP1: 5'-UmsAmsCmUmAmCmAfGmUfUfUfAmGmAmAmGmAmUmUmUmAm-3' (SEQ ID NO: 119) 5'-P1UmsAfsAmAmUmCfUmUmCmUmAmAmAmCfUmGfUmAmGmUmAmsUmsGm-3' (SEQ ID NO: 120) siC5c1-M1P1: 5'-GmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 157) 5'-P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm-3' (SEQ ID NO: 158) siC5c1-M2P1: 5'-GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 159) 5'-P1UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUm-3' (SEQ ID NO: 160) siC5c1-M3P1: 5'-GmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 161) 5'-P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUm-3' (SEQ ID NO: 162) siC5c2-M1P1: 5'-AmGmGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 163) 5'-P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm-3' (SEQ ID NO: 164) siC5c2-M2P1: 5'-AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 165) 5'-P1UmAfUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmGmGm-3' (SEQ ID NO: 166) siC5c2-M3P1: 5'-AmGmGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 167) 5'-P1UmAfUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmGmGm-3' (SEQ ID NO: 168) siC5c1-M1SP1: 5'-GmsGmsAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 169) 5'-P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm-3' (SEQ ID NO: 170) siC5c1-M2SP1: 5'-GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 171) 5'-P1UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmsCmsUm-3' (SEQ ID NO: 172) siC5c1-M3SP1: 5'-GmsGmsAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 173) 5'-P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmsCmsUm-3' (SEQ ID NO: 174) siC5c2-M1SP1: 5'-AmsGmsGmGmAmAmGmGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 175) 5'-P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm-3' (SEQ ID NO: 176) siC5c2-M2SP1: 5'-AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 177) 5'-P1UmsAfsUmUmGmCfUmCfGfGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm-3' (SEQ ID NO: 178) siC5c2-M3SP1: 5'-AmsGmsGmGmAmAmGfGmUfUfAfCmCmGmAmGmCmAmAmUmAm-3' (SEQ ID NO: 179) 5'-P1UmsAfsUmUmGmCfUmCmGmGmUmAmAmCfCmUfUmCmCmCmUmsGmsGm-3' (SEQ ID NO: 180) siC5d1-M1P1: 5'-AmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 217) 5'-P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm-3' (SEQ ID NO: 218) siC5d1-M2P1: 5'-AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 219) 5'-P1UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCm-3' (SEQ ID NO: 220) siC5d1-M3P1: 5'-AmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 221) 5'-P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCm-3' (SEQ ID NO: 222) siC5d2-M1P1: 5'-GmGmAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 223) 5'-P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm-3' (SEQ ID NO: 224) siC5d2-M2P1: 5'-GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 225) 5'-P1UmAfAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmUmGm-3' (SEQ ID NO: 226) siC5d2-M3P1: 5'-GmGmAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 227) 5'-P1UmAfAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmUmGm-3' (SEQ ID NO: 228) siC5d1-M1SP1: 5'-AmsGmsAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 229) 5'-P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm-3' (SEQ ID NO: 230) siC5d1-M2SP1: 5'-AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 231) 5'-P1UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmsCmsCm-3' (SEQ ID NO: 232) siC5d1-M3SP1: 5'-AmsGmsAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 233) 5'-P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmsCmsCm-3' (SEQ ID NO: 234) siC5d2-M1SP1: 5'-GmsGmsAmGmAmAmCmAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 235) 5'-P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm-3' (SEQ ID NO: 236) siC5d2-M2SP1: 5'-GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 237) 5'-P1UmsAfsAmUmUmCfUmGfCfUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm-3' (SEQ ID NO: 238) siC5d2-M3SP1: 5'-GmsGmsAmGmAmAmCfAmGfAfCfAmGmCmAmGmAmAmUmUmAm-3' (SEQ ID NO: 239) 5'-P1UmsAfsAmUmUmCfUmGmCmUmGmUmCmUfGmUfUmCmUmCmCmsUmsGm-3' (SEQ ID NO: 240) siC5e1-M1P1: 5'-CmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 277) 5'-P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm-3' (SEQ ID NO: 278) siC5e1-M2P1: 5'-CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 279) 5'-P1UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCm-3' (SEQ ID NO: 280) siC5e1-M3P1: 5'-CmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 281) 5'-P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCm-3' (SEQ ID NO: 282) siC5e2-M1P1: 5'-GmGmCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 283) 5'-P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm-3' (SEQ ID NO: 284) siC5e2-M2P1: 5'-GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 285) 5'-P1UmUfUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmUmGm-3' (SEQ ID NO: 286) siC5e2-M3P1: 5'-GmGmCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 287) 5'-P1UmUfUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmUmGm-3' (SEQ ID NO: 288) siC5e1-M1SP1: 5'-CmsCmsAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 289) 5'-P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm-3' (SEQ ID NO: 290) siC5e1-M2SP1: 5'-CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 291) 5'-P1UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmsCmsCm-3' (SEQ ID NO: 292) siC5e1-M3SP1: 5'-CmsCmsAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 293) 5'-P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmsCmsCm-3' (SEQ ID NO: 294) siC5e2-M1SP1: 5'-GmsGmsCmCmAmAmGmAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 295) 5'-P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm-3' (SEQ ID NO: 296) siC5e2-M2SP1: 5'-GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 297) 5'-P1UmsUfsUmGmCmAfGmCfGfUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm-3' (SEQ ID NO: 298) siC5e2-M3SP1: 5'-GmsGmsCmCmAmAmGfAmAfGfAfAmCmGmCmUmGmCmAmAmAm-3' (SEQ ID NO: 299) 5'-P1UmsUfsUmGmCmAfGmCmGmUmUmCmUmUfCmUfUmGmGmCmCmsUmsGm-3' (SEQ ID NO: 300) siC5f1-M1P1: 5'-CmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 337) 5'-P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm-3' (SEQ ID NO: 338) siC5f1-M2P1: 5'-CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 339) 5'-P1UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAm-3' (SEQ ID NO: 340) siC5f1-M3P1: 5'-CmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 341) 5'-P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAm-3' (SEQ ID NO: 342) siC5f2-M1P1: 5'-UmAmCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 343) 5'-P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm-3' (SEQ ID NO: 344) siC5f2-M2P1: 5'-UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 345) 5'-P1UmUfUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmAmCm-3' (SEQ ID NO: 346) siC5f2-M3P1: 5'-UmAmCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 347) 5'-P1UmUfUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmAmCm-3' (SEQ ID NO: 348) siC5f1-M1SP1: 5'-CmsCmsAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 349) 5'-P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm-3' (SEQ ID NO: 350) siC5f1-M2SP1: 5'-CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 351) 5'-P1UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmsUmsAm-3' (SEQ ID NO: 352) siC5f1-M3SP1: 5'-CmsCmsAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 353) 5'-P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmsUmsAm-3' (SEQ ID NO: 354) siC5f2-M1SP1: 5'-UmsAmsCmCmAmGmUmAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 355) 5'-P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm-3' (SEQ ID NO: 356) siC5f2-M2SP1: 5'-UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 357) 5'-P1UmsUfsUmCmUmGfGmCfUfUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm-3' (SEQ ID NO: 358) siC5f2-M3SP1: 5'-UmsAmsCmCmAmGmUfAmAfGfCfAmAmGmCmCmAmGmAmAmAm-3' (SEQ ID NO: 359) 5'-P1UmsUfsUmCmUmGfGmCmUmUmGmCmUmUfAmCfUmGmGmUmAmsAmsCm-3' (SEQ ID NO: 360). 23. Use of a siRNA conjugate according to any one of paragraphs. 1-22 for the production of a medicinal product for the treatment and/or prevention of myasthenia gravis. 24. A method of inhibiting the expression of the C5 gene in a hepatocyte, including providing an effective amount of the siRNA conjugate according to any one of claims. 1-22 into contact with the indicated hepatocyte.