SiRNA for inhibiting expression of c3 gene, conjugates thereof and uses thereof

Abstract

The present application relates to siRNA for inhibiting C3 gene expression and conjugates and applications thereof, and belongs to the technical field of biology.The present application provides siRNA for inhibiting C3 gene expression, and experiments prove that the siRNA all have high inhibitory activity on C3 gene expression.The present application also provides modified siRNA for inhibiting C3 expression, and the modification includes methoxy modification, fluorination modification, phosphorothioate group connection, ethylene glycol nucleic acid modification, reverse baseless nucleotide modification, vinyl phosphate modification and deoxyribonucleotide substitution, and experiments prove that the modified siRNA all have good C3 inhibitory effect at different concentrations.

Description

Cross-references to related applications

[0001] This application claims priority to Chinese Patent Application No. 202510177782.4, filed on February 18, 2025, entitled "siRNA for inhibiting C3 and its modifications and applications", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to siRNA and its conjugates for inhibiting C3 gene expression and their applications, belonging to the field of biotechnology. Background Technology

[0003] Complement (C) is a group of heat-sensitive proteins found in the serum and tissue fluid of humans and animals. Upon activation, these proteins possess enzymatic activity and mediate immune responses and inflammatory reactions. Activation of the complement system mediates a series of cellular responses, such as cell lysis, opsonization (antigen-antibody binding), inflammatory responses, and clearance of immune complexes. The complement system is mainly composed of over 30 glycoproteins, including intrinsic components, regulatory proteins, and receptors. These proteins exist either as soluble proteins in the blood or as membrane-associated proteins. Complement activation triggers a sequential cascade of enzymatic reactions (known as the complement activation pathway), leading to the formation of potent anaphylatoxins C3a and C5a. These anaphylatoxins induce a range of physiological responses, from chemotaxis to apoptosis.

[0004] Complement activation is known to occur via three distinct pathways: the alternative pathway, the classical pathway, and the lectin pathway. These pathways involve proteins that exist primarily in inactive proenzyme forms, which are then sequentially cleaved and activated. The classical pathway is typically activated by antibody-antigen complexes or C-reactive protein (CRP), both of which interact with the complement component C1q. Furthermore, the classical pathway can be activated by phosphatidylserine residues present in apoptotic bodies in the absence of immune complexes. The lectin pathway is initiated by mannose-binding lectin (MBL), which binds to complex carbohydrate residues on the surface of pathogens. Activation of either the classical or lectin pathway leads to activation of C3 convertase (C4b2b).

[0005] The alternative pathway is activated on the target surface by the binding of C3b, which is spontaneously generated by the hydrolysis of complement C3. This surface-bound C3b is then recognized by factor B, forming the complex C3bB. In turn, the C3bB complex is cleaved by factor D to produce the active form of the AP, a C3 convertase (C3bBb). Both types of C3 convertases cleave C3 to form C3b. C3b then either binds to more factor B, enhancing complement activation via AP (the so-called alternative or amplified loop), or leads to the formation of an active C5 convertase (C3bBbC3b or C4bC2bC3b), which cleaves C5 and initiates a late event leading to the formation of the membrane attack complex (MAC / C5b-9).

[0006] Aberrant activation of the complement system is a cause of many different disease transmissions and / or the initiation of pathological processes. The deposition of complement C3 in the alternative pathway is one of the important causes of aberrant complement system activation, leading to conditions such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), C3 glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, ischemia-reperfusion injury, and neurodegenerative diseases. However, currently, there are limited therapies available for treating C3-induced complement system aberrant activation-related diseases, which often require time-consuming and invasive procedures and are costly. Therefore, there is an urgent need to find more effective and cost-effective non-invasive treatments for C3-induced complement system aberrant activation-related diseases.

[0007] RNA interference (RNAi) refers to a highly conserved evolutionary phenomenon involving the efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA). Small interfering RNA (siRNA) is a 20-25 nucleotide long double-stranded RNA that mediates the silencing of specific genes through RNA interference (RNAi). In the RNAi pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules. This interference triggers mRNA degradation, thereby inhibiting the expression of specific genes. This specific regulation of gene expression makes siRNA a potential targeted therapeutic agent, specifically regulating the expression of disease-related genes to achieve therapeutic goals. This treatment offers advantages such as high efficiency, low cost, and non-invasiveness. Therefore, developing siRNA that can effectively inhibit C3 expression would be an ideal drug for treating diseases related to C3-induced abnormal activation of the complement system. Summary of the Invention

[0008] To address the aforementioned problems, the present invention provides an siRNA for inhibiting C3 gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 1 to SEQ ID NO. 264 or SEQ ID NO. 529 to SEQ ID NO. 546 by no more than 3 nucleotides; the antisense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 265 to SEQ ID NO. 528 or SEQ ID NO. 547 to SEQ ID NO. 564 by no more than 3 nucleotides.

[0009] In one embodiment of the present invention, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NO.1~SEQ ID NO.264 or SEQ ID NO.529~SEQ ID NO.546; and the nucleotide sequence of the antisense strand of the siRNA is shown in any one of SEQ ID NO.265~SEQ ID NO.528 or SEQ ID NO.547~SEQ ID NO.564.

[0010] In one embodiment of the present invention, at least one nucleotide in the sense strand and / or antisense strand of the siRNA is a modified nucleotide.

[0011] In one embodiment of the present invention, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not cause a significant weakening or loss of the function of the siRNA disclosed herein in inhibiting C3 gene expression.

[0012] In one embodiment of the present invention, the modified nucleotide includes compounds formed by replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, compounds formed by modifying the nucleotide with a thiophosphate group, and / or compounds formed by modifying the bases on the nucleotide.

[0013] In one embodiment of the present invention, the modified nucleotide includes fluorinated nucleotides, methoxylated nucleotides, phosphate-thioester linked nucleotides, glycolic acid (GNA) modified nucleotides, inverse baseless nucleotides modified nucleotides, vinyl phosphate modified nucleotides, deoxyribonucleotide-substituted nucleotides, 2'-O-methoxyethyl (MOE) modified nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 3'-deoxy-nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, and 2'-O-allyl-modified nucleotides. Nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotide-modified nucleotides, aminophosphate-modified nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, open-ring nucleotides (UNA)-modified nucleotides, nucleotides containing methyl phosphate groups, nucleotides containing 5'-phosphate groups, and / or nucleotides containing 5'-phosphate mimics.

[0014] In one embodiment of the present invention, the fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, having the following structure (where Base represents a base): .

[0015] In one embodiment of the present invention, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group, having the following structure (where Base represents a base): .

[0016] In one embodiment of the present invention, the nucleotide linked by the thiophosphate group refers to the thiophosphate group linking two adjacent nucleotides. The thiophosphate group has the following structure: ; The nucleotide linked to the thiophosphate group has the following structure (where Base represents a base): .

[0017] In one embodiment of the present invention, the glycol nucleic acid (GNA) modified nucleotide is a polymer similar to DNA or RNA, but its "backbone" is composed of repeating glycerol units linked by phosphodiester bonds; the glycol nucleic acid (GNA) modified nucleotide includes A (GNA), G (GNA), C (GNA), U (GNA) and / or T (GNA); The A(GNA) has the following structure: ; The G(GNA) has the following structure: ; The C(GNA) has the following structure: ; The U(GNA) has the following structure: ; The T(GNA) has the following structure: .

[0018] In one embodiment of the present invention, the nucleotide modified with a reverse abase nucleotide refers to a nucleotide coupled with a reverse abase nucleotide (invAb), wherein the reverse abase nucleotide has the following structure: .

[0019] In one embodiment of the present invention, the 5' position of the sugar ring of the vinyl phosphate-modified nucleotide is modified with vinyl phosphate; the vinyl phosphate-modified nucleotide includes VPAms, VPUms, VPGms and / or VPCms (VP ​​represents (E)-vinyl phosphate). The VPAms have the following structure: ; The VPUms have the following structure: ; The VPGms has the following structure: ; The VPCms has the following structure: .

[0020] In one embodiment of the present invention, the deoxyribonucleotide-substituted nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a hydrogen atom, which has the following structure (where Base represents a base): .

[0021] In one embodiment of the present invention, the nucleotide modified with 2'-O-methoxyethyl (MOE) refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with 2'-O-methoxyethyl (MOE), which has the following structure (where Base represents a base): .

[0022] In one embodiment of the present invention, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 8, and 9 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5, 7, 8, 9, and 15 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 8, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 8, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 9, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 4, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides.

[0023] In one embodiment of the present invention, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, 9, and 15 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 8, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 8, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 9, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 4, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides.

[0024] In one embodiment of the present invention, the methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and in the sense strand, the nucleotides without fluorination, without ethylene glycol nucleic acid (GNA) modification, and without deoxyribonucleotide substitution are all methoxylated nucleotides; in the antisense strand, the nucleotides without fluorination, without ethylene glycol nucleic acid modification, and without deoxyribonucleotide substitution are all methoxylated nucleotides.

[0025] In one embodiment of the invention, the nucleotide modified with an inverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 3' end of the first nucleotide in the positive strand is attached to an inverse abase-free nucleotide (invAb group); and / or, The inverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the penultimate nucleotide of the positive strand is attached to an inverse abase-free nucleotide (invAb group).

[0026] In one embodiment of the invention, the nucleotide modified with inverse abase nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 3' end of the first nucleotide of the positive strand is attached to an inverse abase nucleotide (invAb group), and the 5' end of at least the penultimate nucleotide of the positive strand is attached to an inverse abase nucleotide (invAb group).

[0027] In one embodiment of the invention, the vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the antisense strand is attached to a trans vinyl phosphate group (VP group).

[0028] In one embodiment of the invention, the vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of the first nucleotide of the antisense strand is attached to a trans vinyl phosphate group (VP group).

[0029] In one embodiment of the invention, the deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at the 5th position of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 10 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 16 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 12 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 11, 12, and 13 of the antisense strand are deoxyribonucleotide-substituted nucleotides; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides.

[0030] In one embodiment of the invention, the deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotide at position 5 of the antisense strand is the deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 10 of the antisense strand is the deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 16 of the antisense strand is the deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 12 of the antisense strand is the deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 11, 12, and 13 of the antisense strand are deoxyribonucleotide-substituted nucleotides; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and the nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides in the direction from the 5' end to the 3' end.

[0031] In one embodiment of the present invention, the nucleotide modified with ethylene glycol nucleic acid (GNA) is located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at least the 7th position of the antisense strand is ethylene glycol nucleic acid.

[0032] In one embodiment of the present invention, the nucleotide modified with ethylene glycol nucleic acid (GNA) is located in the antisense and sense strands of the nucleotide sequence, and the nucleotide at position 7 of the antisense strand is ethylene glycol nucleic acid in the direction from the 5' end to the 3' end.

[0033] In one embodiment of the invention, the nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1, and positions 2 to 1 ... The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group, and the nucleotide at position 1-1-3 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group; at least the nucleotides at positions 1-2, 2-3, 1-1-2-3, and 1-1-2-2-3 of the antisense strand are linked by a phosphate-thioester group; or, The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the reverse non-base nucleotide by a phosphate-thioester link, the nucleotide at position 1 to the reverse non-base nucleotide is linked by a phosphate-thioester link, and the nucleotide at position 1 to position 2 to position 3 of the antisense strand is linked to the reverse non-base nucleotide by a phosphate-thioester link, and the nucleotide at position 1 to position 2 to position 3 of the antisense strand is linked to the reverse non-base nucleotide by a phosphate-thioester link.

[0034] In one embodiment of the present invention, the nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1, and positions 2 to 1 ... The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the inverted abasic nucleotide via a phosphate-thioester group, and the nucleotide at position 1-3 of the sense strand is linked to the inverted abasic nucleotide via a phosphate-thioester group; the nucleotides at positions 1-2, 2-3, 1-1-2, and 1-1-2 of the antisense strand are linked via phosphate-thioester groups; or, The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence. Furthermore, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the reverse non-base nucleotide by a thiophosphate group, the nucleotide at position 1 to the reverse non-base nucleotide is linked by a thiophosphate group, and the nucleotides at positions 1 to 2 to 3 to 4 to 5 to 6 to 5 are linked by a thiophosphate bond. The nucleotides at positions 1 to 2, 2 to 3, 1 to 2 to 3 to 4 to 5 to 6 to 5 to 6 to 7 to 6 to 6 to 7 to 6 to 6 to 7 to 6 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 6 to 7 to 7 to 8 ...

[0035] The present invention also provides an siRNA conjugate for inhibiting C3 gene expression, the siRNA conjugate containing the above-mentioned siRNA and a conjugating group conjugated to the siRNA.

[0036] In one embodiment of the present invention, the conjugation group comprises a ligand formed from a targeting molecule or a derivative thereof; the targeting molecule comprises galactose and / or N-acetylgalactosamine (GalNAc).

[0037] In one embodiment of the present invention, the conjugating group is N-glucose amino acid (N-Acetylgalactosamine, GalNAc).

[0038] In one embodiment of the present invention, the conjugation site of the siRNA and the conjugation group is located at the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the internal sequence of the siRNA sense strand, the 5' end of the siRNA antisense strand, or the internal sequence of the siRNA antisense strand.

[0039] In one embodiment of the present invention, the conjugation site of the siRNA and the conjugating group is located at the 3' end of the positive strand of the siRNA.

[0040] In one embodiment of the present invention, the conjugation group is conjugated to the 3' end of the siRNA positive strand via a phosphodiester bond.

[0041] In one embodiment of the present invention, the siRNA conjugate has the following structure: .

[0042] The present invention also provides a recombinant plasmid expressing the above-mentioned siRNA.

[0043] In one embodiment of the present invention, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a bacteriophage vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector, hybrid virus vector, baculovirus vector, herpes simplex virus vector, or lentivirus vector; the non-viral vector includes a plasmid vector.

[0044] In one embodiment of the present invention, the recombinant plasmid is prepared by: designing shRNA based on siRNA; and ligating the shRNA with a linearized vector to obtain the recombinant plasmid.

[0045] The present invention also provides a host cell whose genome integrates the above-mentioned siRNA; or, whose genome integrates the above-mentioned antisense oligonucleotide; or, whose host cell carries the above-mentioned recombinant plasmid.

[0046] In one embodiment of the present invention, the host cell includes fungi, bacteria, plant cells and / or animal cells.

[0047] The present invention also provides the use of the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned recombinant plasmid or the above-mentioned host cell in the preparation of medicaments for the prevention and / or treatment of diseases, wherein the diseases are related to abnormal activation of the complement system caused by C3.

[0048] In one embodiment of the present invention, the C3-induced complement system abnormal activation-related diseases include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), C3 glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, ischemia-reperfusion injury and / or neurodegenerative diseases.

[0049] The present invention also provides a medicament for the prevention and / or treatment of a disease related to abnormal activation of the complement system caused by C3; the medicament comprises the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned recombinant plasmid or the above-mentioned host cell.

[0050] In one embodiment of the present invention, the C3-induced complement system abnormal activation-related diseases include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), C3 glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, ischemia-reperfusion injury and / or neurodegenerative diseases.

[0051] In one embodiment of the present invention, the drug further comprises other bioactive substances that inhibit the complement system; the bioactive substances that inhibit the complement system include small molecules, antibodies, siRNA, ASO, or modified gene therapy products.

[0052] In one embodiment of the present invention, the bioactive substance that inhibits the complement system includes an antibody that inhibits C5 expression (such as Ravulizumab), an siRNA that inhibits C5 expression (such as cemdisiran), a small molecule that inhibits CFB expression (such as Iptacopan), and / or an siRNA that inhibits CFB expression (such as ARO-CFB).

[0053] In one embodiment of the present invention, the drug further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include drug carriers, pH buffers, lyophilization protectants, osmotic pressure regulators, excipients, stabilizers and / or preservatives.

[0054] In one embodiment of the present invention, the drug carrier includes magnetic nanoparticles (such as Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethyl ethylene phosphate). One or more of phosphate), PPEEA and poly(2-dimethylaminoethylmethacrylate) (PDMAEMA) and its derivatives.

[0055] In one embodiment of the present invention, the pH buffer includes a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5 to 8.5 and / or a phosphate buffer with a pH of 5.5 to 8.5.

[0056] In one embodiment of the present invention, the protective agent includes one or more of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0057] In one embodiment of the present invention, the osmotic pressure regulator includes sodium chloride and / or potassium chloride.

[0058] The technical solution of this invention has the following advantages: This invention provides an siRNA for inhibiting C3 gene expression. The siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 1~SEQ ID NO. 264 or SEQ ID NO. 529~SEQ ID NO. 546 by no more than 3 nucleotides; the antisense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 265~SEQ ID NO. 528 or SEQ ID NO. 547~SEQ ID NO. 564 by no more than 3 nucleotides. Experiments have shown that the siRNA exhibits high inhibitory activity against C3. Therefore, the siRNA shows great promise in the preparation of drugs for the prevention and / or treatment of diseases related to abnormal activation of the complement system caused by C3.

[0059] Furthermore, at least one nucleotide in the sense and / or antisense strands of the siRNA is a modified nucleotide; the modification includes methoxy modification, fluorination modification, thiophosphate linkage, glycol nucleic acid modification, reverse abasic nucleotide modification, vinyl phosphate modification, and deoxyribonucleotide substitution. Experiments have shown that the modified siRNA exhibits good C3 inhibition effects at different concentrations. Therefore, the modified siRNA shows great promise in the preparation of drugs for the prevention and / or treatment of diseases related to C3-induced abnormal activation of the complement system. Attached Figure Description

[0060] Figure 1 Effects of different siRNA conjugates on C3 gene expression in mice (single-dose experiment, dose 3 mg / kg, duration 14 days).

[0061] Figure 2 Effects of different siRNA conjugates on C3 gene expression in rats (single-dose experiment, dose 1.5 mg / kg, duration 14 days).

[0062] Figure 3 Effects of different siRNA conjugates on C3 gene expression in humanized mice (single-dose experiment, dose 3 mg / kg, duration 14 days).

[0063] Figure 4 Effects of different siRNA conjugates on C3 gene expression in humanized mice (single-dose experiment, dose 3 mg / kg, duration 35 days). Detailed Implementation

[0064] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0065] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0066] In the following embodiments, C3 mRNA refers to mRNA having the sequences shown in GeneBank registration numbers Homo C3 NM_000064.4, Mouse C3 NM_009778.3, cynomolgus monkey C3 XM_005587719.4, and Rat C3 NM_016994.2. Further, unless otherwise specified, the term "target gene" as used in this disclosure refers to the gene that transcribes the above-mentioned C3 mRNA, and the term "target mRNA" refers to the above-mentioned C3 mRNA.

[0067] Unless otherwise specified, the reagents and culture media used in the following examples are commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are performed in accordance with the methods described in Molecular Biology (4th Edition) (Alexander McLennan et al., 2019).

[0068] The experimental cells used in the following examples are HepG2 cells, purchased from the Cell Bank of the Chinese Academy of Sciences.

[0069] The experimental animals were female C57BL / 6J mice (4-6 weeks old) and male SD rats (6-8 weeks old), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed in the SPF-grade animal facility of Suzhou Gemma Gene Co., Ltd. Animals were kept under a 12-hour light-dark cycle and had free access to food and water. Experiments began after one week of acclimatization. The use and handling of the experimental animals complied with the requirements of the Animal Management Committee of Suzhou Gemma Gene Co., Ltd. regarding experimental animals and animal welfare.

[0070] The siRNAs involved in the following examples are siRNA sequences synthesized via phosphoramide solid-phase synthesis.

[0071] In the examples described below, when transfecting cells with siRNA targeting the C3 gene, siRNA conjugates, or siRNA or siRNA conjugates as negative controls, Lipo3000 or RNAiMAX (purchased from Invitrogen) were used as transfection reagents, and the specific procedures were performed according to the manufacturer's instructions. For qPCR detection, HiScript III RT SuperMix for qPCR (purchased from Vazyme) was used as the reverse transcription reagent, and the specific procedures were performed according to the manufacturer's instructions.

[0072] Example 1: A siRNA for inhibiting C3 This embodiment provides an siRNA for inhibiting C3, the nucleotide sequence of which is designed based on the target mRNA, as shown in Table 1.

[0073] Table 1. siRNAs and their sequences that inhibit C3 gene expression

[0074] Example 2: A modified siRNA and siRNA conjugate for inhibiting C3 This embodiment provides a modified siRNA and siRNA conjugate for inhibiting C3. The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 8, and 9 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and 2 and 3. Similarly, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions. Alternatively, The modified siRNA is based on the siRNA of Example 1, with the following changes: nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated at the 2' position, and nucleotides at other positions are methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first and penultimate nucleotides are linked by a phosphate thioester group. Nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated at the 2' position. The 5th nucleotide is replaced with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on). The 7th nucleotide is ethylene glycol nucleic acid. Nucleotides modified with acid (GNA), where the nucleotides at other positions are methoxylated at the 2' position, and the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester bonds; or, The modified siRNA is based on the siRNA of Example 1, with the following changes: nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated at the 2' position, and nucleotides at other positions are methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first and penultimate nucleotides are linked by a phosphate thioester group. Nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated at the 2' position. The 5th nucleotide is replaced with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on). The 7th nucleotide is ethylene glycol nucleic acid. Nucleotides modified with GNA (acid, GNA), with methoxy groups at the 2' position, and a trans-vinyl phosphate group (VP group) attached to the 5' end of the nucleotide at position 1. Phosphophosphate bonds connect the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. The modified siRNA is based on the siRNA of Example 1, with the 5th, 7th, 8th, 9th, and 15th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A thiophosphate bond connects the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides. Similarly, the 2nd, 6th, 8th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. A thiophosphate bond connects the 1st and 2nd nucleotides, the 2nd and 3rd nucleotides, the penultimate and penultimate nucleotides, and the penultimate and penultimate nucleotides. Alternatively, The modified siRNA is based on the siRNA of Example 1, wherein the 5th, 7th, 8th, 9th, and 15th nucleotides of the sense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position; the 1st and 2nd nucleotides, and the 2nd and 3rd nucleotides are linked by a thiophosphate bond; the 2nd, 6th, 8th, 14th, and 16th nucleotides of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position; a trans-vinyl phosphate group (VP group) is attached to the 5' end of the 1st nucleotide; and the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides are linked by a thiophosphate bond; or, The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and 2 and 3. Similarly, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions. Alternatively, The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and 2 and 3. On the antisense strand, the nucleotides at positions 2, 6, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A trans-vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions. Alternatively, The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, the penultimate nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, and the penultimate and penultimate nucleotides are linked by a phosphate thioester bond. The 2nd, 6th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. Phosphothioester bonds are used to link the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate and penultimate nucleotides are linked by a thiophosphate bond. The 2nd, 8th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. Thiophosphate bonds are used to link the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, the penultimate nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, and the penultimate and penultimate nucleotides are linked by a phosphate thioester bond. The 2nd, 9th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. Phosphothioester bonds are used to link the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate and penultimate nucleotides are linked by a thiophosphate bond. The 2nd, 10th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. Thiophosphate bonds are used to link the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the positive strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group. The nucleotides at the penultimate and penultimate positions are linked by a phosphate thioester bond. The nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are fluorinated at the 2' position. The nucleotide at position 10 is substituted with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide replaces uracil ribonucleotide, and so on). The nucleotides at other positions are methoxylated at the 2' position. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by a phosphate thioester bond; or... The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the positive strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group. The nucleotides at the penultimate and penultimate positions are linked by a phosphate thioester bond. The nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand are fluorinated at the 2' position. The nucleotide at position 16 is substituted with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide replaces uracil ribonucleotide, and so on). The nucleotides at other positions are methoxylated at the 2' position. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by a phosphate thioester bond; or... The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the positive strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group. The nucleotides at the penultimate and penultimate positions are linked by a phosphate thioester bond. The nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated at the 2' position. The nucleotide at position 12 is substituted with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide replaces uracil ribonucleotide, and so on). The nucleotides at other positions are methoxylated at the 2' position. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by a phosphate thioester bond; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the positive strand having a 2' fluorination, and the nucleotides at other positions having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group. The nucleotides at positions 2, 6, 10, 14, and 16 of the sense chain are fluorinated at the 2' position, the nucleotide at position 12 is substituted with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on), the nucleotides at other positions are methoxylated at the 2' position, the 5' end of the nucleotide at position 1 is attached to a trans-vinyl phosphate group (VP group), and the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds; or, The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the positive strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group. The nucleotides at the penultimate and penultimate positions are linked by a phosphate thioester bond. The nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are fluorinated at the 2' position. The nucleotide at position 12 is substituted with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide replaces uracil ribonucleotide, and so on). The nucleotides at other positions are methoxylated at the 2' position. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by a phosphate thioester bond; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the positive strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group. The first and second-to-last nucleotides are linked by a phosphate thioester bond. The nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated at the 2' position. The nucleotides at positions 11, 12, and 13 are deoxyribonucleotide substitutions (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide replaces uracil ribonucleotide, and so on). The nucleotides at other positions are methoxylated at the 2' position. The nucleotides at positions 1 and 2, 2 and 3, the second-to-last and second-to-last, and the second-to-last and third-to-last are linked by phosphate thioester bonds; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate and penultimate nucleotides are linked by a thiophosphate bond. The 2nd, 4th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. Thiophosphate bonds are used to link the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a thiophosphate group. The 2nd, 4th, 12th, 14th, and 16th nucleotides of the antisense strand have a 2' fluorination, and the other nucleotides have a 2' methoxylation. A trans-vinyl phosphate group (VP group) is attached to the 5' end of the 1st nucleotide. Thiophosphate bonds connect the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides; or... The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 9, 10, and 11 of the positive strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group, and the penultimate nucleotide and the reverse abasic nucleotide are linked by a... The phosphate thioester group is linked, with the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand having a 2' fluorination, and the nucleotides at positions 5 and 7 having a deoxyribonucleotide substitution (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on). The nucleotides at other positions have a 2' methoxy group. Phosphothioester bonds connect the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the nucleotide at position 1, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the nucleotide from the penultimate position. The nucleotides at positions 1 and 2 are linked by a phosphate thioester group, and the nucleotides from the penultimate position are also linked by a phosphate thioester group. The 2' position of the antisense strand... Nucleotides at positions 10, 12, 14, 16, 18, and 20 are fluorinated at the 2' position. Nucleotides at positions 5 and 7 are deoxyribonucleotides (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on). Nucleotides at other positions are methoxylated at the 2' position. A trans-vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. Nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds.

[0075] The siRNA conjugate consists of modified siRNA and a GalNAc group conjugated to the modified siRNA; the siRNA conjugate has the following structure (double helix structure represents siRNA, and the linker is attached to the 3' end of the siRNA's positive strand): .

[0076] Modified siRNAs and siRNA conjugates are shown in Tables 2-5.

[0077] Table 2. Modified siRNAs that suppress C3 gene expression and their sequences

[0078] In Table 2, uppercase letters C, G, U, and A represent ribonucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; and lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate groups.

[0079] Table 3. Modified siRNAs, siRNA conjugates, and their sequences that inhibit C3 gene expression

[0080] Table 4. Modified siRNAs, siRNA conjugates, and their sequences that inhibit C3 gene expression

[0081] Table 5. Modified siRNAs, siRNA conjugates, and their sequences that inhibit C3 gene expression

[0082] In Tables 3-5, uppercase letters C, G, U, and A represent ribonucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by a thiophosphate group; string (d) indicates that the nucleotide adjacent to the left of this string is replaced by a deoxyribonucleotide; string (GNA) indicates that the nucleotide adjacent to the left of this string is a glycerol nucleic acid (GNA) modified nucleotide; string (invAb) indicates that the nucleotide adjacent to the left or right of this string is linked to an inverse abase nucleotide (invAb group); string VP indicates that the nucleotide adjacent to the right of this string is a vinyl phosphate modified nucleotide.

[0083] Experiment 1: Verification of the activity of siRNA used to inhibit C3 in HepG2 cells This experimental example provides a validation experiment for the activity of siRNA inhibiting C3 in HepG2 cells. The experimental procedure is as follows: HepG2 cells were seeded into MEM medium (Gibco, catalog number 11095-080) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone) and 1% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, catalog number 15140122) and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h. After culture, the HepG2 cells were digested with trypsin (Gibco, catalog number 25200-072). After digestion, the cells were rinsed with PBS buffer and then resuspended in MEM medium to obtain a cell concentration of 3 × 10⁶ cells / year. 5 Cell suspension of cells / mL; Different siRNAs were diluted separately using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilutions containing different siRNAs. 25 μL of opti-MEM was mixed with 0.25 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilutions. 25 μL of each siRNA dilution was then mixed with the transfection reagent dilutions and incubated at room temperature (25℃) for 15 min to obtain transfection solutions containing different siRNAs. Cell suspension was seeded into 96-well plates at a seeding rate of 50 μL / well. A transfection reagent control group (MOCK) and an siRNA experimental group were set up in each well, with 3 replicates per group. After setup, 50 μL of transfection buffer containing different siRNAs was added to the wells of the siRNA experimental group (hhC3_442M group received transfection buffer containing hC3_442M, hC3_443M group received transfection buffer containing hC3_443M, and so on; the final concentration of siRNA in the wells was 0.01 nM, 0.1 nM, 1 nM, or 5 nM, respectively, and the final siRNA concentration was adjusted using Opti-MEM dilution). 50 μL of transfection buffer without any siRNA was added to the wells of the transfection reagent control group (MOCK). Cells were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h for transfection. After transfection, the liquid in the wells was discarded, the cells were collected, and total RNA was extracted from the cells in each well using a magnetic bead method total RNA extraction kit (purchased from Suzhou Genegene Co., Ltd., catalog number E31008) according to the method described in the kit instructions. The experimental procedure was performed using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number R323-01) according to the product instructions. A 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions to reverse transcribe total RNA from cells. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 37°C for 15 min, then at 85°C for 5 s. 80 μL of DEPC water was added to each reverse transcription reaction system to obtain a solution containing cDNA. For each reverse transcription reaction system, 4 μL of the above-mentioned cDNA-containing solution was used as a template. Using the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02), 20 μL of qPCR reaction system was prepared on an ice box according to Table 6. Primer 1 and Primer 2 are the PCR primer sequences for amplifying the target gene C3 and the internal reference gene GAPDH, respectively (as shown in Table 7). Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and amplification was performed using a three-step method. The amplification program was: 95℃ pre-denaturation for 10 min, then 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene C3 and internal reference gene GAPDH. Product W was then subjected to 95℃ for 15 s, 60℃ for 15 s, and 60℃ for 30 s. After incubation at 95℃ for 1 min and 95℃ for 15 s, the melting curves of the target gene C3 and the internal reference gene GAPDH in product W were collected by real-time fluorescence quantitative PCR instrument to obtain the Ct values ​​of the target gene C3 and the internal reference gene GAPDH. The relative quantification of the target gene C3 in each test group was performed using the comparison Ct(ΔΔCt) method, as follows: ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group); ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group); ΔCt(test group) = ΔCt(test group) - ΔCt(control group average); ΔCt(control group) = ΔCt(control group) - ΔCt(control group average); Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) for each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value; Using the control group as a baseline, the expression level of C3 mRNA in the test group was normalized, and the expression level of C3 mRNA in the control group was defined as 100%. The relative expression level of C3 mRNA in the test group was 2. -ΔΔCt(测试组) ×100%; C3 mRNA inhibition rate in test group = 1 - relative expression level of C3 mRNA in test group; The C3 mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the saline control group. The data are expressed as a percentage relative to the saline control group and presented as the mean plus the standard deviation. The experimental results are shown in Tables 8 to 13.

[0084] siRNA concentration gradients were set at 10 nM, 1 nM, 0.33 nM, 0.11 nM, 0.037 nM, 0.0123 nM, and 0.0041 nM. Following the method described above for detecting the relative expression level of C3 mRNA, the effects of gradient concentrations of siRNA on the relative expression level of C3 mRNA were examined. Then, using Grapdhpad software, IC50 curves for different siRNAs were fitted using nonlinear regression. The experimental results are shown in Table 13.

[0085] As shown in Tables 8-12, the modified siRNA and siRNA conjugates in Example 2 all exhibited good C3 inhibition effects at different concentrations.

[0086] Table 6. DNA Amplification Reaction System

[0087] Table 7. Primer Information

[0088] Table 8. Inhibitory levels of different modified siRNAs on C3 mRNA in HepG2 cells

[0089]

[0090]

[0091]

[0092]

[0093] Table 9. Inhibitory levels of different modified siRNAs on C3 mRNA in HepG2 cells

[0094]

[0095] Table 10. Inhibitory levels of different modified siRNAs on C3 mRNA in HepG2 cells

[0096]

[0097] Table 11. Inhibitory levels of different modified siRNAs on C3 mRNA in HepG2 cells

[0098]

[0099] Table 12. Median inhibition concentration (IC50) of different siRNA conjugates on C3 mRNA in HepG2 cells.

[0100] Table 13. Inhibitory levels of different siRNAs on C3 mRNA in HepG2 cells

[0101]

[0102]

[0103] Experiment Example 2: Verification of the activity of siRNA used to inhibit C3 in mice This experimental example provides a validation experiment for the activity of siRNA inhibiting C3 in mice. The experimental procedure is as follows: Experiment 1: C57BL / 6J mice were divided into a saline control group and an siRNA experimental group, with 3 mice in each group. Mice in the siRNA experimental group were administered a single subcutaneous injection of 3 mg / kg of different siRNAs (in 200 μL of saline solution), while mice in the saline control group were injected with an equal volume of saline. On day 14 after administration, the mice were sacrificed, liver samples were collected and flash-frozen in liquid nitrogen, liver mRNA was extracted, and analyzed using RT-qPCR to evaluate the inhibitory effect of siRNA on C3 expression in mice.

[0104] Experiment 2: SD rats were divided into a saline control group and an siRNA experimental group, with 3 rats in each group. Rats in the siRNA experimental group received a single subcutaneous administration of 1.5 mg / kg of different siRNAs (in 200 μL of saline solution), while rats in the saline control group received an equal volume of saline solution. On day 14 after administration, the rats were sacrificed, liver samples were collected and flash-frozen in liquid nitrogen, and liver mRNA was extracted and analyzed using RT-qPCR to evaluate the inhibitory effect of siRNA on C3 expression in rats.

[0105] Experiment 3: The experiment of Experiment 1 was repeated using hC3 humanized mice to evaluate the inhibitory effect of siRNA on C3 expression in humanized mice.

[0106] Experiment 4: hC3 humanized mice were divided into a saline control group and an siRNA experimental group, with 3 mice in each group. Mice in the siRNA experimental group were administered a single subcutaneous injection of 3 mg / kg of different siRNAs (in 200 μL of saline solution), while mice in the saline control group were injected with an equal volume of saline. On day 35 post-administration, the mice were sacrificed, liver samples were collected and flash-frozen in liquid nitrogen, liver mRNA was extracted, and analyzed using RT-qPCR to evaluate the inhibitory effect of siRNA on C3 expression in humanized mice.

[0107] The detection steps for RT-qPCR are as follows: Step 1: RNA extraction 1) Take 20 mg of mouse liver tissue, add 1 mL of Trizol Lysis Buffer (purchased from Life Technology, catalog number 410701), grind and lyse the tissue, transfer the completely dissolved mixture to an RNase-free 1.5 mL centrifuge tube; shake vigorously for about 15 seconds to fully lyse the tissue cells, and let stand at room temperature (25℃) for 5 minutes; 2) Carefully open the tube cap and add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., catalog number 20140925); shake vigorously for 20 seconds, let stand at room temperature (25℃) for 3 minutes; centrifuge at 12000×g for 20 minutes at 4℃. 3) After centrifugation, carefully remove the centrifuge tubes to the centrifuge tube rack, transfer the supernatant to a new 2.0 mL centrifuge tube, add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., product number 20210802) to the supernatant, and mix by inverting. 4) Take a purification column with a collection tube (purchased from VWI, catalog number 11822AG0627), add 700 μL of the mixture from step 3), let stand for 2 min; centrifuge at 10000×g for 1 min at 4℃, discard the filtrate; repeat the above steps with the remaining mixture. 5) Add 700 μL of 80% (v / v) ethanol to the purification column, centrifuge at 10000×g for 1 min at 4℃, and discard the filtrate; 6) Add 700 μL of 80% (v / v) ethanol to the purification column, centrifuge at 10000×g for 1 min at 4℃, and discard the filtrate; 7) Centrifuge the purification column at 4℃, 10000×g for 2 min (empty). 8) After centrifugation, carefully remove the purification column with the collection tube (if there is liquid in the collection tube, please be careful not to splash the liquid onto the purification column), discard the collection tube, put the purification column into a new 1.5mL centrifuge tube, add 100μL LEPC water to the purification column, let it stand at room temperature (25℃) for 2min; centrifuge at 4℃, 10000×g for 1min. 9) Collect the RNA solution from step 8) for subsequent experiments; Step 2: RNA reverse transcription The experimental procedure was performed using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number R323-01) according to the product instructions. A 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions to reverse transcribe total RNA from cells. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 37°C for 15 min, then at 85°C for 5 s. 80 μL of DEPC water was added to each reverse transcription reaction system to obtain a solution containing cDNA. Step 3: RT-qPCR procedure, Example 1. See the experimental results below. Figures 1-4 .

[0108] Figures 1-4 The experimental results in Example 2 showed that the modified siRNA and siRNA conjugates in Example 2 had good inhibitory effects on C3 in mice at different application concentrations and durations of treatment.

[0109] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A siRNA for inhibiting C3 gene expression, characterized in that, The siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA contains at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO.1~SEQ ID NO.264 or SEQ ID NO.529~SEQ ID NO.546 by no more than 3 nucleotides; the antisense strand of the siRNA contains at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO.265~SEQ ID NO.528 or SEQ ID NO.547~SEQ ID NO.564 by no more than 3 nucleotides.

2. The siRNA as described in claim 1, characterized in that, At least one nucleotide in the sense strand and / or antisense strand of the siRNA is a modified nucleotide.

3. The siRNA as described in claim 2, characterized in that, The modified nucleotides include compounds formed by replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, compounds formed by modifying the thiophosphate group of a nucleotide, and / or compounds formed by modifying the bases on a nucleotide.

4. The siRNA according to any one of claims 2 to 3, characterized in that, The modified nucleotides include fluorinated nucleotides, methoxylated nucleotides, phosphate-thioester linked nucleotides, ethylene glycol-modified nucleotides, inverse abase-free nucleotides, vinyl phosphate-modified nucleotides, deoxyribonucleotide-substituted nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine nucleotides, 3'-deoxy-nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, abase-free nucleotides, and 2'-amino- - Modified nucleotides, 2'-O-allyl- modified nucleotides, 2'-C-alkyl- modified nucleotides, 2'-hydroxy- modified nucleotides, 2'-O-alkyl- modified nucleotides, morpholinyl nucleotide modified nucleotides, aminophosphate-modified nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl alcohol-modified nucleotides, cyclohexenyl-modified nucleotides, open-ring nucleotide modified nucleotides, nucleotides containing methyl phosphate groups, nucleotides containing 5'-phosphate groups, and / or nucleotides containing 5'-phosphate mimics.

5. The siRNA as described in claim 4, characterized in that, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 8, and 9 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5, 7, 8, 9, and 15 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 8, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 8, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 9, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 4, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or, The fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, 16, 18, and 20 of the antisense strand are fluorinated nucleotides.

6. The siRNA as described in claim 4 or 5, characterized in that, The methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence. In the sense strand, the nucleotides that are not fluorinated, not ethylene glycol-modified, and not substituted with deoxyribonucleotides are all methoxylated nucleotides. In the antisense strand, the nucleotides that are not fluorinated, not ethylene glycol-modified, and not substituted with deoxyribonucleotides are all methoxylated nucleotides.

7. The siRNA according to any one of claims 4 to 6, characterized in that, The nucleotide modified with a reverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 3' end of the first nucleotide of the positive strand is attached to a reverse abase-free nucleotide; and / or, The reverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the penultimate nucleotide of the positive strand is attached to a reverse abase-free nucleotide.

8. The siRNA according to any one of claims 4 to 7, characterized in that, The vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the antisense strand is attached to a trans vinyl phosphate group.

9. The siRNA according to any one of claims 4 to 8, characterized in that, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 5 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 10 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 16 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 12 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 11, 12, and 13 of the antisense strand are deoxyribonucleotide-substituted nucleotides; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides.

10. The siRNA according to any one of claims 4 to 9, characterized in that, The glycolic acid-modified nucleotide is located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th nucleotide in the antisense strand is glycolic acid-modified.

11. The siRNA according to any one of claims 4 to 10, characterized in that, The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by phosphate-thioester groups; and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1, and positions 2 to 1 ... The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group, and the nucleotide at position 1-1-3 of the sense strand is linked to the inverted abasic nucleotide by a phosphate-thioester group; at least the nucleotides at positions 1-2, 2-3, 1-1-2-3, and 1-1-2-2-3 of the antisense strand are linked by a phosphate-thioester group; or, The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 1 of the sense strand is linked to the reverse non-base nucleotide by a phosphate-thioester link, the nucleotide at position 1 to the reverse non-base nucleotide is linked by a phosphate-thioester link, and the nucleotide at position 1 to position 2 to position 3 of the antisense strand is linked to the reverse non-base nucleotide by a phosphate-thioester link, and the nucleotide at position 1 to position 2 to position 3 of the antisense strand is linked to the reverse non-base nucleotide by a phosphate-thioester link.

12. A siRNA conjugate for inhibiting C3 gene expression, characterized in that, The siRNA conjugate contains the siRNA as described in any one of claims 1 to 11 and a conjugating group conjugated to the siRNA.

13. A recombinant plasmid, characterized in that, The recombinant plasmid expresses the siRNA according to any one of claims 1 to 11.

14. A host cell, characterized in that, The host cell genome is integrated with the siRNA according to any one of claims 1 to 11; or, the host cell carries the recombinant plasmid according to claim 13.

15. The use of the siRNA according to any one of claims 1 to 11, the siRNA conjugate according to claim 12, the recombinant plasmid according to claim 13, or the host cell according to claim 14 in the preparation of a medicament for the prevention and / or treatment of a disease, wherein the disease is a disease related to abnormal activation of the complement system caused by C3.

16. A medicine for preventing and / or treating a disease, characterized in that, The disease is a C3-induced complement system abnormal activation-related disease; the drug contains siRNA as described in any one of claims 1 to 11, siRNA conjugate as described in claim 12, recombinant plasmid as described in claim 13, or host cell as described in claim 14.

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