CFB inhibitor composition and use thereof

Abstract

The present invention relates to the technical field of nucleic acid drugs, and particularly provides a double-stranded oligonucleotide for inhibiting complement factor B (CFB) gene expression. The double-stranded oligonucleotide comprises a sense strand and an antisense strand; the sense strand contains at least fifteen consecutive nucleotides in any one sequence shown as SEQ ID NO: 1-SEQ ID NO: 255 in Table 1 or a nucleotide sequence which differs from the at least fifteen consecutive nucleotides in respect of not more than three nucleotides; and / or, the antisense strand contains at least fifteen consecutive nucleotides in any one sequence shown as SEQ ID NO: 256-SEQ ID NO: 510 in Table 1 or a nucleotide sequence which differs from the at least fifteen consecutive nucleotides in respect of not more than three nucleotides. The double-stranded oligonucleotide can inhibit CFB gene expression in cells, thereby achieving the effects of relieving, treating and / or preventing diseases or disorders mediated by abnormal CFB gene expression.

Description

CFB inhibitor composition and its application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310938654.8 filed with the State Intellectual Property Office of China on July 28, 2023, entitled “CFB inhibitor composition and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present disclosure belongs to the technical field of nucleic acid drugs, and in particular relates to an RNAi composition for inhibiting complement factor B (CFB) gene expression, such as a double-stranded ribonucleic acid (dsRNA) composition and its application. Background Art

[0004] The complement system is part of the host's innate immune system and is composed of multiple proteins that exist in the blood as soluble or membrane-associated proteins. Inappropriate activation of the complement system contributes to the proliferation and / or initiation of lesions in many diseases. Complement factor B is a key component of the alternative pathway of complement activation and circulates in the blood as a single polypeptide chain. Following activation of the alternative pathway, factor B is cleaved by complement factor D, producing the non-catalytic chain Ba and the catalytic subunit Bb. The active subunit Bb is a serine protease that binds to C3b to form the alternative pathway C3 convertase. C3 convertase catalyzes the formation of C3 into C3a and C3b. C3b, in turn, binds to C3 convertase, regulated by CFB, leading to a cascade of enzymatic reactions, including the generation of C5 convertase, which activates the membrane attack pathway, leading to the formation of a series of membrane attack complexes (MACs), triggering a variety of physiological responses, including inflammation and even apoptosis.

[0005] The complement system regulates immunity and plays an important role in normal inflammatory responses, but dysregulation of the complement system can lead to complement dysregulation-related diseases. Inhibition of excessive or dysregulated complement activation can treat or alleviate related diseases or conditions.

[0006] Therefore, there is a need in the art for treating diseases or disorders associated with complement activation by modulating complement factor B activity.

[0007] Summary of the Invention

[0008] The present disclosure provides a complement factor B (CFB) inhibitor, comprising a double-stranded oligonucleotide, a double-stranded RNAi agent, and a pharmaceutical composition comprising the same; the present disclosure provides a method for treating, preventing, or alleviating a disease associated with dysregulation of the alternative complement pathway in a subject by administering a complement factor B (CFB) inhibitor. The present disclosure also provides a method for inhibiting CFB expression by administering a CFB-specific inhibitor (double-stranded RNAi agent) to a subject.

[0009] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0010] In a first aspect, the present disclosure provides a double-stranded oligonucleotide (dsRNA) for inhibiting complement factor B (CFB) gene expression. The double-stranded oligonucleotide is capable of inhibiting CFB gene expression in mammals, including humans, monkeys, rats, or mice. The double-stranded oligonucleotide can inhibit CFB gene expression in cells in vitro and in vivo.

[0011] The double-stranded oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides of any one of SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or the antisense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides;

[0012] Preferably, the sense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1.

[0013] The sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region; the substantially complementary means that the sense strand and the antisense strand have no more than 3 nucleotide mismatches in the double-stranded region.

[0014] In a second aspect, the present disclosure provides a double-stranded RNAi agent for inhibiting complement factor B (CFB) gene expression, characterized in that the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, wherein the substantially complementary region refers to a mismatch of no more than 3 nucleotides between the sense strand and the antisense strand in the double-stranded region, and the double-stranded region is represented by the following formula (I):

[0015] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0016] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (I),

[0017] Wherein, SS represents the sense strand, and AS represents the antisense strand;

[0018] All nucleotides of the sense strand and the antisense strand are modified nucleotides;

[0019] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy modified nucleotide;

[0020] The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides, wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino.

[0021] Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10;

[0022] Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

[0023] In a third aspect, the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of complement factor B (CFB), the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1. Wherein, each strand is no more than 25 nucleotides in length, and both the sense strand and the antisense strand are modified nucleotides. The modified nucleotides comprise modified nucleotides selected from 2'-O-methyl modification and 2'-fluoro modification, and the sense strand comprises 1-3 phosphorothioate internucleotide bonds; wherein the modified nucleotides in the antisense strand comprise modified nucleotides selected from 2'-O-methyl modification, 2'-O-methoxyethyl modification and 2'-fluoro modification, wherein the antisense strand comprises 2-5 phosphorothioate internucleotide bonds; and wherein the sense strand is conjugated to a GalNAc ligand at the 3'-end.

[0024] In a fourth aspect, the present disclosure provides a use of a double-stranded RNAi agent in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by a complement factor B gene.

[0025] In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising the double-stranded RNAi agent described in the present disclosure and pharmaceutically optional excipients.

[0026] In a sixth aspect, the present disclosure provides a method for inhibiting complement factor B (CFB) expression in cells in vitro, the method comprising:

[0027] (a) contacting the cell with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition thereof; and

[0028] (b) maintaining the cells produced in step (a) for a period of time sufficient to achieve degradation of mRNA transcripts of the complement factor B (CFB) gene, thereby inhibiting the expression of the complement factor B (CFB) gene in the cells, wherein the expression of the complement factor B (CFB) gene is inhibited by at least 70%.

[0029] In a seventh aspect, the present disclosure provides use of the double-stranded RNAi agent or a pharmaceutical composition thereof in the preparation of a medicament for treating a subject suffering from a complement factor B (CFB)-related disorder.

[0030] In an eighth aspect, the present disclosure provides use of the double-stranded RNAi agent in the preparation of a medicament for treating a subject suffering from a disorder associated with complement factor B (CFB) disorder, wherein the medicament is subcutaneously administered to the subject, optionally, wherein the subject is a human or other mammal.

[0031] In a ninth aspect, the present disclosure provides a kit, characterized in that the kit comprises the double-stranded RNAi agent or its pharmaceutical composition described in the present disclosure.

[0032] In a tenth aspect, the present disclosure provides a method for inhibiting complement factor B gene expression, characterized in that the method comprises administering to a subject a double-stranded RNAi agent or a pharmaceutical composition thereof as described herein. In some embodiments, the expression of complement factor B is inhibited by at least 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the inhibition of complement factor B expression reduces the level of complement factor B protein in the subject's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0033] In an eleventh aspect, the present disclosure provides a method for alleviating, treating and / or preventing diseases or conditions mediated by complement factor B, characterized in that the method comprises administering the double-stranded RNAi agent or a pharmaceutical composition thereof described in the present disclosure to a subject.

[0034] The diseases or conditions mediated by complement factor B include diseases related to the mRNA level of complement factor B gene expression.

[0035] The complement factor B-related disease is selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), asthma, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, psoriasis, dermatomyositis bullous pemphigoid, atypical hemolytic uremic syndrome, Shiga toxin-infected Escherichia coli-associated hemolytic uremic syndrome, myasthenia gravis, neuromyelitis optica, dense deposit disease, C3 neuropathy, age-related macular degeneration, antineutrophil cytoplasmic antibody-associated vasculitis, myocardial infarction, and sepsis.

[0036] Optionally, the disease or disorder includes, but is not limited to, renal disease, systemic lupus erythematosus and related diseases, macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0037] Optionally, the renal disease comprises C3 glomerulopathy, lupus nephritis, IgA nephropathy, diabetic nephropathy, membranous nephropathy and polycystic kidney disease.

[0038] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0040] FIG1 shows the inhibitory activity of target genes in primary mouse liver cells after administration of double-stranded RNAi agents in Experiment 3.

[0041] FIG2 shows the inhibitory activity of target genes in C57BL / 6J mice after administration of double-stranded RNAi agents in Experiment 4. FIG.

[0042] FIG3 shows the inhibitory activity of target genes in C57BL / 6J mice after administration of different ligands and modified double-stranded RNAi agents in Experiment 5. ... DETAILED DESCRIPTION

[0043] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure. The technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, not all. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present disclosure.

[0044] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0045] In a first aspect, the present disclosure provides a double-stranded oligonucleotide (dsRNA) for inhibiting complement factor B (CFB) gene expression. The double-stranded oligonucleotide is capable of inhibiting CFB gene expression in mammals, including humans, monkeys, rats, or mice. The double-stranded oligonucleotide can inhibit CFB gene expression in cells in vitro and in vivo.

[0046] The double-stranded oligonucleotide includes a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides of any one of SEQ ID NO:1-SEQ ID NO:255 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or, the antisense strand comprises at least 15 consecutive nucleotides of any one of SEQ ID NO:256-SEQ ID NO:510 in Table 1, or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region; the substantial complementarity means that the sense strand and the antisense strand have no more than 3 nucleotide mismatches in the double-stranded region.

[0047] In some optional embodiments, the sense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any one of the sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1.

[0048] In some optional embodiments, the sense strand comprises a nucleotide sequence that differs by no more than 1 or 2 nucleotides from any one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 1 or 2 nucleotides from any one of the sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1.

[0049] In some optional embodiments, the sense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 1 nucleotide from any of the sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1.

[0050] In some specific embodiments of the present disclosure, the sense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and / or the antisense strand comprises any one of the nucleotide sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1.

[0051] In some optional embodiments, the sense strand or the antisense strand comprises any sense strand or antisense strand selected from the duplexes shown in the following numbers in Table 1: RN011001, RN011005, RN011006, RN011007, RN011018, RN011021, RN011022, RN011033, RN011034, RN011035, RN011038, RN011052, RN011076, RN011097, RN011098, RN011101, RN011102, RN011103, RN011104, RN011106, RN011107, RN01108 N011109, RN011113, RN011114, RN011117, RN011123, RN011124, RN011125, RN011140, RN01115, RN011151, RN011152, RN011156, RN011160, RN011 171, RN011183, RN011193, RN011194, RN011199, RN011201, RN011202, RN011233, RN011238, RN011241, RN011242, RN011243, RN011254, RN011255.

[0052] In some embodiments, the sense strand or the antisense strand comprises a duplex nucleotide sequence selected from the group consisting of RN011255.

[0053] In some optional embodiments, the double-stranded oligonucleotide comprises one or more selected from the duplex group shown in the following numbers in Table 1: RN011001, RN011005, RN011006, RN011007, RN011018, RN011021, RN011022, RN011033, RN011034, RN011035, RN011038, RN011052, RN011076, RN011097, RN011098, RN011101, RN011102, RN011103, RN011104, RN011106, RN011107, RN01108 11109, RN011113, RN011114, RN011117, RN011123, RN011124, RN011125, RN011140, RN01115, RN011151, RN011152, RN011156, RN011160, RN0111 71. RN011183, RN011193, RN011194, RN011199, RN011201, RN011202, RN011233, RN011238, RN011241, RN011242, RN011243, RN011254, RN011255;

[0054] In some embodiments, the double-stranded oligonucleotide comprises a duplex nucleotide sequence selected from the group consisting of RN011255.

[0055] In some alternative embodiments, each nucleotide of the double-stranded oligonucleotide is an unmodified or modified nucleotide.

[0056] In some alternative embodiments, at least one nucleotide in the double-stranded oligonucleotide is modified.

[0057] In some alternative embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand of the double-stranded oligonucleotide are modified nucleotides.

[0058] In some optional embodiments, the modified nucleotides are selected from 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more nucleotides substituted with OR; wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino.

[0059] In some optional embodiments, the modified nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n OR one or more of the nucleotides substituted.

[0060] In some specific embodiments of the present disclosure, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 3, and / or the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 3.

[0061] In some optional embodiments, the length of the antisense strand and the sense strand is 17-25 nucleotides.

[0062] In some optional embodiments, at least one of the antisense strand and the sense strand comprises a 3'-end overhang of 1-2 nucleotides.

[0063] In a second aspect, the present disclosure provides a double-stranded RNAi agent for inhibiting complement factor B (CFB) gene expression, characterized in that the double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, wherein the substantially complementary region refers to a mismatch of no more than 3 nucleotides between the sense strand and the antisense strand in the double-stranded region, and the double-stranded region is represented by the following formula (I):

[0064] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0065] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (I),

[0066] Wherein, SS represents the sense strand, and AS represents the antisense strand;

[0067] All nucleotides of the sense strand and the antisense strand are modified nucleotides;

[0068] Each of the Ns independently represents a modified nucleotide: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy modified nucleotide;

[0069] The Xs are each independently a 2'-O-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) nOR substituted modified nucleotides, wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino;

[0070] Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10;

[0071] Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

[0072] In some optional embodiments, the N is selected from 2'-O-methyl modified nucleotides and 2'-fluoro-modified nucleotides.

[0073] In some alternative embodiments, the nucleotide X is a 2'-O-methyl modified nucleotide or a 2'-O-methoxyethyl modified nucleotide.

[0074] In some alternative embodiments, the duplex comprises at least one 2'-O-methoxyethyl modification or 2'-O(CH2) n OR modified nucleotides.

[0075] In some alternative embodiments, the double-stranded RNAi agent comprises a duplex consisting of the following modified sense and antisense strands, in the direction from 5' end to 3' end:

[0076] The justice chain is: GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmAm, or

[0077] GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmsAm;

[0078] The antisense strand is: UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfAmAfUmCmCmsUmsCm, or

[0079] UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfAmAfUmCmCmsUmsCm, or

[0080] UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfA(moe)AfUmCmCmsUmsCm, or

[0081] UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfA(moe)AfUmCmCmsUmsCm, or

[0082] UmsUfsAmUmAmGfAmAmAfAmsCmCmCmAfA(moe)AfUmCmCmsUmsCm.

[0083] In some optional embodiments, the nucleotides of the double-stranded RNAi sense chain and / or antisense chain are all modified nucleotides, and, wherein, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides; and optionally, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense chain are 2'-fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0084] In some optional embodiments, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

[0085] In some optional embodiments, in the double-stranded RNAi agent, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7-10 in the nucleotide sequence of the positive strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides.

[0086] In some optional embodiments, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

[0087] In some optional embodiments, in the double-stranded RNAi agent, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the return strand are 2'-fluorinated modified nucleotides, and any one nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides.

[0088] In some optional embodiments, in the double-stranded RNAi agent, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides;

[0089] Furthermore, positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and: any nucleotide selected from positions 9, 10, 11, or 12 is a 2'-fluoro-modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl-modified nucleotide, and the remaining positions are 2'-O-methyl-modified nucleotides.

[0090] In some optional embodiments, the nucleotides of the sense strand and / or the antisense strand are all modified nucleotides; and the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(4):

[0091] (1) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the return strand are 2'-fluorinated nucleotides, and the remaining positions are 2'-O-methyl modified nucleotides;

[0092] (2) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the return strand are 2'-fluorinated nucleotides, and the remaining positions are 2'-O-methyl modified nucleotides;

[0093] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the return strand are 2'-fluorinated nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides;

[0094] (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 in the nucleotide sequence of the return strand are 2'-fluorinated nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides.

[0095] In some alternative embodiments, the double-stranded RNAi agent further comprises a ligand.

[0096] In some alternative embodiments, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi agent.

[0097] In some alternative embodiments, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives connected by a linker, and the linker is selected from a monovalent, divalent or trivalent linker.

[0098] In some optional embodiments, the ligand structure is as follows:

[0099] Wherein, * represents the attachment site of the oligonucleotide molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L and Y are defined as follows: Z is independently selected from hydroxyl or sulfhydryl; p is independently selected from 1, 2 or 3; q is independently selected from 1, 2 or 3; each R3 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; L is C1-C 20 The alkylene chain, or one or more C atoms in the chain, is substituted by any substituent selected from O, S, NH or -NH-C(O)-; Y is independently selected from NH, O or S.

[0100] In some optional embodiments, the specific structure of the ligand is as follows:

[0101] In some alternative embodiments, the 3' end of the sense strand in the double-stranded RNAi agent is covalently coupled to a ligand.

[0102] In some alternative embodiments, the double-stranded RNAi agent of the present disclosure comprises a conjugate represented by the following formula:

[0103] Wherein, Nu represents a double-stranded oligonucleotide molecule;

[0104] The sense strand of the double-stranded oligonucleotide molecule comprises any modified sense strand nucleotide sequence shown in Table 3; and / or, the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 3;

[0105] The ligand is linked to the 3' end of the sense strand.

[0106] In some alternative embodiments, the double-stranded RNAi agent of the present disclosure comprises a duplex selected from the group consisting of RZ011304, RZ011305, RZ011306, and RZ011307.

[0107] In some alternative embodiments, the sense strand in the double-stranded RNAi agent is:

[0108] 5'-GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmAm_(CR01008×3)-3', or

[0109] 5'-GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmsAm_(CR01008×3)-3';

[0110] The antisense strand is:

[0111] 5'-UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfAmAfUmCmCmsUmsCm-3', or

[0112] 5'-UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfAmAfUmCmCmsUmsCm-3', or

[0113] 5'-UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfA(moe)AfUmCmCmsUmsCm-3', or

[0114] 5'-UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfA(moe)AfUmCmCmsUmsCm-3', or

[0115] 5'-UmsUfsAmUmAmGfAmAmAfAmsCmCmCmAfA(moe)AfUmCmCmsUmsCm-3';

[0116] where Am, Cm, Gm, and Um are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine phosphate, and 2'-O-methyluridine phosphate, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; A(moe) is 2'-O-methoxyethyladenosine-3'-phosphate, and s is phosphorothioate;

[0117] Wherein CR01008×3 is a ligand with the following structure:

[0118] In some optional embodiments, the sense strand and / or the antisense strand further comprises a 3' and / or 5' extension or overhang of 1-3 nucleotides in length.

[0119] In some optional embodiments, the sense strand of the double-stranded region is 17-21 nt long, and the backsense strand is 19-23 nt long. Preferably, the double-stranded region is 19 to 23 nucleotide pairs long; or, the double-stranded region is 19 to 21 nucleotide pairs long.

[0120] In some alternative embodiments, the sense strand and / or the backsense strand of the RNAi agent independently comprises one or more phosphorothioate internucleoside linkages.

[0121] In some alternative embodiments, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends, or the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends.

[0122] In some alternative embodiments, at least one strand of the double-stranded region of the RNAi agent comprises a 3' overhang of 1-2 nucleotides.

[0123] In some alternative embodiments, the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein each strand is 14-25 nucleotides, and the antisense strand comprises a region complementary to a portion of the mRNA encoding CFB (SEQ ID NO: 526), ​​wherein the double-stranded region is represented by the following Formula II:

[0124] SS:5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3'

[0125] AS:3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (II)

[0126] wherein SS represents the sense strand, AS represents the antisense strand, and the SS strand can be conjugated to a ligand; each of the Ns independently represents a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-deoxy-modified nucleotide; and each of the Xs independently represents a 2'-O-methoxyethyl modified nucleotide.

[0127] wherein said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0, and 0≤q'+r'≤2;

[0128] Furthermore, there is at least one fluorinated-modified nucleotide in (N)a, and the 16th position of the sense strand, counted from the 5'-end, is a fluorinated-modified nucleotide; there is at least one fluorinated-modified nucleotide in (N)b, and the 14th position of the sense strand, counted from the 5'-end, is a fluorinated-modified nucleotide; there are at least two fluorinated-modified nucleotides in (N)c, and positions 2 and 6 of the sense strand, counted from the 5'-end, are both fluorinated-modified nucleotides; and, counted from the 5'-end, the first four nucleotides of (N)b' contain at least two fluorinated-modified nucleotides.

[0129] In some optional embodiments, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain of Formula II are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense chain are 2'-fluorinated modified nucleotides, and: any nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides.

[0130] In some optional embodiments, the double-stranded RNAi agent comprises a group consisting of duplexes selected from the group consisting of the following duplexes: RZ011304, RZ011305, RZ011306, and RZ011307.

[0131] In a third aspect, the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of complement factor B (CFB), the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1. Wherein, each strand is no more than 25 nucleotides in length, and both the sense strand and the antisense strand are modified nucleotides. The modified nucleotides comprise modified nucleotides selected from 2'-O-methyl modification and 2'-fluoro modification, and the sense strand comprises 1-3 phosphorothioate internucleotide bonds; wherein the modified nucleotides in the antisense strand comprise modified nucleotides selected from 2'-O-methyl modification, 2'-O-methoxyethyl modification and 2'-fluoro modification, wherein the antisense strand comprises 2-5 phosphorothioate internucleotide bonds; and wherein the sense strand is conjugated to a GalNAc ligand at the 3'-end.

[0132] In some alternative embodiments, the double-stranded RNAi agent comprises a duplex selected from the group consisting of the duplexes shown in the following numbers: RZ011304, RZ011305, RZ011306, and RZ011307.

[0133] In a fourth aspect, the present disclosure provides a use of a double-stranded RNAi agent in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by a complement factor B gene.

[0134] In some alternative embodiments, the disease or condition comprises kidney disease, systemic lupus erythematosus and related diseases, macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis.

[0135] In some alternative embodiments, the renal disease comprises C3 glomerulopathy, lupus nephritis, IgA nephropathy, diabetic nephropathy, membranous nephropathy, and polycystic kidney disease.

[0136] In a fifth aspect, the present disclosure provides a pharmaceutical composition comprising the double-stranded RNAi agent described in the present disclosure and pharmaceutically optional excipients.

[0137] In some optional embodiments, the double-stranded RNAi agent of the present disclosure can be administered in an unbuffered solution or a buffer, wherein the unbuffered solution is saline or water, and the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof; alternatively, wherein the buffer is phosphate-buffered saline (PBS).

[0138] In a sixth aspect, the present disclosure provides a method for inhibiting complement factor B (CFB) expression in cells in vitro, the method comprising:

[0139] (a) contacting the cell with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition thereof; and

[0140] (b) maintaining the cells produced in step (a) for a period of time sufficient to achieve degradation of mRNA transcripts of the complement factor B (CFB) gene, thereby inhibiting the expression of the complement factor B (CFB) gene in the cells, wherein the expression of the complement factor B (CFB) gene is inhibited by at least 70%.

[0141] In a seventh aspect, the present disclosure provides use of the double-stranded RNAi agent or a pharmaceutical composition thereof in the preparation of a medicament for treating a subject suffering from a complement factor B (CFB)-related disorder.

[0142] In an eighth aspect, the present disclosure provides use of the double-stranded RNAi agent in the preparation of a medicament for treating a subject suffering from a disorder associated with complement factor B (CFB) disorder, wherein the medicament is subcutaneously administered to the subject, optionally, wherein the subject is a human or other mammal.

[0143] In some alternative embodiments, the double-stranded RNAi agent is administered at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg.

[0144] In some alternative embodiments, the double-stranded RNAi agent is administered subcutaneously or intravenously.

[0145] In a ninth aspect, the present disclosure provides a kit comprising the double-stranded RNAi agent or a pharmaceutical composition thereof as described in the present disclosure.

[0146] In a tenth aspect, the present disclosure provides a method for inhibiting complement factor B gene expression, the method comprising administering to a subject a double-stranded RNAi agent or a pharmaceutical composition thereof as described herein. In some embodiments, the expression of complement factor B is inhibited by at least 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the inhibition of complement factor B expression reduces the level of complement factor B protein in the subject's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0147] In an eleventh aspect, the present disclosure provides a method for alleviating, treating and / or preventing a disease or condition mediated by complement factor B, comprising administering the double-stranded RNAi agent of the present disclosure or a pharmaceutical composition thereof to a subject.

[0148] In some alternative embodiments, the complement factor B-mediated disease or disorder includes a disease associated with the mRNA level of complement factor B gene expression.

[0149] In some optional embodiments, the complement factor B-related disease is selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), asthma, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, psoriasis, dermatomyositis bullous pemphigoid, atypical hemolytic uremic syndrome, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome, myasthenia gravis, neuromyelitis optica, dense deposit disease, C3 neuropathy, age-related macular degeneration, antineutrophil cytoplasmic antibody-associated vasculitis, myocardial infarction, and sepsis.

[0150] In some optional embodiments, the disease or condition includes but is not limited to kidney disease, systemic lupus erythematosus and related diseases, macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria and rheumatoid arthritis; further preferably, the kidney disease includes C3 glomerulopathy, lupus nephritis, IgA nephropathy, diabetic nephropathy, membranous nephropathy and polycystic kidney disease.

[0151] The experimental data of the examples show that the double-stranded RNAi agent or its pharmaceutical composition provided by the present disclosure can effectively regulate the expression level of the complement factor B gene, and thus can effectively treat and / or prevent diseases or symptoms associated with dysregulated complement factor B gene expression.

[0152] In some optional embodiments, the pharmaceutical composition can be administered by any suitable means, such as in the form of an injection or infusion solution (e.g., subcutaneously, intramuscularly, or intravenously), and the administration of the double-stranded oligonucleotide or the pharmaceutical composition includes, but is not limited to, single administration or multiple administration. The dosage range is 0.1 mg / kg to 100 mg / kg, 0.5 mg / kg to 50 mg / kg, such as 3 mg / kg, 10 mg / kg, or 33 mg / kg.

[0153] It is well known to those skilled in the art that modified nucleotide groups can be introduced into the double-stranded RNAi agents of the present invention by using nucleoside monomers having corresponding modifications. Methods for preparing nucleoside monomers having corresponding modifications and methods for introducing modified nucleotide groups into double-stranded RNAi agents are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.

[0154] [definition]

[0155] In this document, unless otherwise specified, capital letters A (adenine), U (uracil), G (guanine), C (cytosine), and T (thymine, which also represents uracil in the sequence table) represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; (moe) indicates that the nucleotide adjacent to the left of the combination logo is a 2'-O-methoxyethyl-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group.

[0156] L96 represents a structural portion linked to Nu (double-stranded oligonucleotide) represented by the following formula.

[0157] As used herein, "fluorinated nucleotides" or "2'-fluorinated nucleotides" refer to nucleotides in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and "non-fluorinated nucleotides" refer to nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. The "methoxy-modified nucleotides" or "2'-O-methoxyethyl-modified nucleotides" refer to nucleotides in which the 2' hydroxyl group of the ribose group is replaced by a methoxy group or a methoxyethyl group. "Methoxy-modified nucleotides" are also recorded as 2'-OMe or 2'-O-methyl-modified nucleotides, which can be used interchangeably. "2'-O-methoxyethyl-modified nucleotides" are also recorded as 2'-MOE modifications. 2'-deoxynucleotides refer to nucleotides in which the 2' position of the ribose group is hydrogen.

[0158] As used herein, a double-stranded oligonucleotide is composed of two chains, wherein the chain that binds to the target sequence is called the antisense strand or guide strand, and the other chain is called the sense strand or passenger strand. The term "antisense strand" refers to a chain of a double-stranded oligonucleotide that includes a region that is completely or substantially complementary to the target sequence. The term "sense strand" refers to a chain of a double-stranded oligonucleotide that includes a region that is substantially complementary to the region of the antisense strand as defined herein. The term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target sequence. In the case where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the interior or terminal regions of the molecule. As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. As used herein, double-stranded oligonucleotides and siRNA can be used interchangeably.

[0159] As used herein, the expressions "duplex," "duplex region," and "double-stranded region" are used interchangeably and have the meanings known to those skilled in the art, i.e., a double-stranded region in a double-stranded nucleic acid molecule where the sense strand and the antisense strand are completely or substantially complementary to each other.

[0160] As used herein, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases of the other strand in a complementary manner.

[0161] As used herein, unless otherwise specified, "substantially reverse complementary" or "substantially complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than one base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there are no base mismatches between the two nucleotide sequences.

[0162] The term "complement factor B" (used interchangeably with the term "CFB") refers to genes and polypeptides well known in the art, known in the art as: AHUS, BF, CFAB, BFD, FB, GBG, FBI12, B-factor, Properdin, H2-Bf, Glycine-rich beta glycoprotein, C3 proaccelerator, Properdin factor 2B, C3 activator precursor, PBF2, Glycine-rich beta glycoprotein, C3 / C5 convertase, and EC 3.4.21, and EC 3.4.21.473. The term "CFB" includes: human CFB, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:189181756; mouse CFB, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:218156288 and GI:218156290; rat CFB, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:218156284; and chimpanzee CFB, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:57114201. The term "CFB" also includes cynomolgus macaque CFB, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. GI:544428919. Additional examples of CFB mRNA sequences are readily available, for example, using GenBank, UniProt, the Online Mendelian Inheritance in Man (OMIM), and the Macaque Genome Sequencing Project website.

[0163] Exemplary CFB nucleotide sequences of the present disclosure are listed in Tables 1-2. The term "CFB" also refers to naturally occurring DNA sequence variations of the CFB gene. It also refers to single nucleotide polymorphisms within the CFB gene. Many sequence variations within the CFB gene have been identified and can be discovered, as is known in the art.

[0164] The terms "RNAi," "iRNA," "RNAi agent," "RNAi reagent," "RNA interference agent," and "RNA inhibitor" are used interchangeably herein to refer to molecules or agents that contain RNA and can mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. It is well known in the art that RNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). In one embodiment, the RNAi agents of the present disclosure include single-stranded or double-stranded RNA that interacts with a target RNA sequence (e.g., a CFB, C3, or C9 target mRNA sequence) to direct the cleavage of the target RNA. Therefore, in one aspect, the present disclosure relates to the term "siRNA" which can also be used to refer to the RNAi described above.

[0165] In some embodiments, the "RNAi" used in the compositions, uses, and methods of the present disclosure is double-stranded RNA, and the "RNAi agent" includes the double-stranded RNA, and the "RNAi agent" can refer to a "double-stranded RNAi agent", a "double-stranded RNA (dsRNA) molecule", a "dsRNA agent", a "siRNA agent", or a "dsRNA agent".

[0166] "dsRNA" or "siRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to a target RNA (e.g., CFB, C3 or C9 gene).

[0167] In addition, as used in this specification, "RNAi reagents" or "RNAi agents" can include ribonucleotides with chemical modifications and / or ligands; RNAi reagents can include substantial modifications at multiple nucleotides. The term "modified nucleotides" refers to nucleotides that independently have modified sugar moieties, modified internucleotide linkages, and / or modified nucleobases. Thus, the term modified nucleotides encompasses substitutions, additions, or removals of, for example, functional groups or atoms of internucleotide linkages, sugars, or nucleobases. Modifications suitable for use in the agents of the present disclosure include all types of modifications disclosed herein or known in the art. As used in siRNA molecules, any such modification can be encompassed by "RNAi agent."

[0168] The term "nucleotide overhang" or "overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA (e.g., dsRNA). For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. The one or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang can be present on the 5' end, the 3' end, or both ends of the antisense or sense strand of the dsRNA.

[0169] The term "ligand" generally refers to any compound or molecule that can be covalently or otherwise chemically bound to a biologically active substance (such as an oligonucleotide). In certain embodiments, a ligand can interact directly or indirectly with another compound, such as a receptor. The receptor that interacts with the ligand can be present on the cell surface, or alternatively can be an intracellular and / or intercellular receptor. The interaction of the ligand with the receptor can result in a biochemical reaction, or can be simply a physical interaction or binding.

[0170] The term "administer" generally refers to introducing the disclosed pharmaceutical formulation into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting cells, organs, or tissues with the drug can be employed. The administration may include, but is not limited to, intravenous, intraarterial, intranasal, intraperitoneal, intramuscular, subcutaneous, or oral administration. The daily dose may be divided into one, two, or more doses in a suitable form for administration at one, two, or more times during a certain time period.

[0171] The term "contacting" generally refers to the contacting of two or more different types of substances in any order, in any manner, and for any duration. Contact can occur in vivo, ex vivo, or in vitro. In some embodiments, it can refer to direct contact of a RNAi agent or composition of the present disclosure with a cell or tissue. In other embodiments, the term refers to indirect contact of a RNAi agent or composition of the present disclosure with a cell or tissue.

[0172] The term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, improvement, prevention and / or treatment of a disease, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), livestock (dogs and cats), farm animals (horses, cows, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adult subjects. Subjects include animal disease models. In the present disclosure, the terms "include", "comprising", "having", "can", "containing" and their variants are generally intended to be open transitional phrases, terms or words that do not exclude the possibility of additional behavior or structure. The term "consisting of..." generally means that no other components (or similarly, features, integers, steps, etc.) can be present. Unless the context clearly dictates otherwise, nouns of undefined number also include plural indicators.

[0173] The mRNA sequence encoding CFB of the present invention (SEQ ID NO: 526) is as follows:

[0174] In the context of the present disclosure, unless otherwise specified, the siRNA sequences used in the present disclosure were commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the synthesis of PCR primers used in the present disclosure was commissioned to Sangon Biotech (Shanghai) Co., Ltd.; the human liver cancer cell line HepG2 used in the present disclosure was purchased from Wuhan Prosai Life Science Co., Ltd.; the experimental animal C57BL / 6J mice used in the present disclosure were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.

[0175] In the context of this disclosure, unless otherwise stated, the real-time PCR detection data of the activity experiments involved in this disclosure are all calculated using the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0176] ΔCt(test group) = Ct(test group target gene) - Ct(test group reference gene)

[0177] ΔCt(control group) = Ct(control group target gene) - Ct(control group internal reference gene)

[0178] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0179] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0180] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%.

[0181] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (Test group) × 100%

[0182] Test group target gene mRNA inhibition rate = 100% - test group target gene mRNA relative expression level

[0183] In the context of this disclosure, unless otherwise stated, in vivo activity experimental data are presented as The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0184] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0185] The double-stranded oligonucleotide (dsRNA) sequences described in the present disclosure are shown in Table 1.

[0186] Table 1 Unmodified double-stranded oligonucleotide sequence list

[0187] Preparation of ligands

[0188] Preparation Example 1 Preparation of CR01008 ligand

[0189] (1.1) Synthesis of compound CR01008

[0190] The synthetic route of compound CR01008 is as follows:

[0191] (1.1.1) Synthesis of Compound 2

[0192] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of a 45.3 wt% aqueous KOH solution was added dropwise. After the addition was complete, the mixture was stirred at 25°C for 30 minutes, then heated to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to slurry it, and the mixture was filtered to obtain compound 2 (9 g, yield 78.9%) as a white solid. MS-ESI (m / z) = 260 [M+H] +.

[0193] (1.1.2) Synthesis of compound 3

[0194] Compound 2 (9 g, 1 eq) prepared in step (1.1.1) was dissolved in 70 mL of 1,4-dioxane. A 4 M solution of hydrogen chloride in 1,4-dioxane (45 mL) was added, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0195] (1.1.3) Synthesis of compound 5

[0196] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) prepared in step (1.1.2) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under a nitrogen atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and subjected to reverse purification (22 vol% acetonitrile in water) to obtain compound 5 (1.78 g, 64.4% yield) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0197] (1.1.4) Synthesis of Compound 6

[0198] Compound 5 (1.54 g, 1.0 eq) prepared in step (1.1.3) was dissolved in 15 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and reverse-phase purified (60% acetonitrile in water) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0199] (1.1.5) Synthesis of compound CR01008

[0200] Compound 6 (1.08 g, 1.0 eq) prepared in step (1.1.4) was dissolved in 20 ml of anhydrous dichloromethane. DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added, respectively. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. After reverse purification (72 vol% acetonitrile in water), the mixture was dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0201] 1 H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,6H),1.37-1.17(m,5H),1.60-1.40(m,6H),1.68-1.62(m,1H),1.80(s,3H), 1.80(s,3H),1.92(s,3H),2.02(s,5H),2.13(s,3H),2.71(t,J=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,1H),3.36(s,1H),3.58- 3.39(m,3H),3.69-3.60(m,2H),3.75(s,7H),3.90(dt,J=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,1H),5.78(s,1H),6.93-6.87(m,4H),7.35-7.21(m,7H),7.44-7.37(m,2H),7.66(d,J=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0202] (1.2) Synthesis of compound CR01008Z

[0203] Compound CR01008Z was obtained by linking compound 6, which was used to synthesize compound CR01008, to a solid phase support CPG.

[0204] The synthetic route of compound CR01008Z is as follows:

[0205] (1.2.1) Synthesis of compound 9

[0206] Compound 6 (500 mg) prepared in step (1.1.4) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg), and TEA (226.2 mg) were added. The atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0207] (1.2.2) Synthesis of compound CR01008Z

[0208] Compound 9 (50 mg), aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (1.2.1) were added to a 20 ml sample vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0209] Cap1 and Cap2 are capping reagents, Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0210] Preparation Example 2 Preparation of (CR01008) × 3 ligand

[0211] By the phosphoramidite nucleic acid solid phase synthesis method, the solid phase support compound (CR01008Z) described in Preparation Example 1 was used as the starting cycle to connect the compounds (CR01008) that were not connected to the solid phase support one by one.

[0212] Each compound connection involves four steps: deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given below:

[0213] The compound CR01008 not bound to a solid support was separately prepared into a 0.1 M solution using acetonitrile.

[0214] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0215] The conditions for each coupling reaction were the same. The coupling reaction conditions were: a temperature of 25°C, a molar ratio of the compound attached to the solid support to the compound not attached to the solid support of 1:10, a molar ratio of the compound attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0216] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the compound attached to the solid support was 1:1:1.

[0217] The conditions for each oxidation or sulfidation reaction were identical. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; sulfidation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of sulfidation agent to the compound attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (water:pyridine volume ratio, 1:9).

[0218] Three clusters of CR01008 (denoted as (CR01008)×3) were obtained through the above method.

[0219] The structural formula of the three-cluster CR01008 is as follows:

[0220] Preparation of double-stranded RNAi agents

[0221] Example 1 Preparation of double-stranded RNAi agent with L96 as ligand

[0222] Step 1: L96-PS

[0223] Compound L96-PS was purchased from Ailiying Pharmaceutical Group (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is as follows:

[0224] Wherein, PS represents polystyrene resin solid phase carrier.

[0225] Step 2: Synthesis of the Sense Strand

[0226] Using phosphoramidite solid-phase nucleic acid synthesis, the L96-PS compound attached to a solid support is used as the initial cycle, and nucleoside monomers are attached one by one in the 3'-5' direction according to the nucleotide sequence. Each nucleoside monomer attachment involves four steps: deprotection, coupling, capping, and oxidation or sulfurization.

[0227] Using the phosphoramidite nucleic acid solid-phase synthesis method, the solid-phase support compound is initially linked to nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given below:

[0228] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0229] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0230] The conditions for each coupling reaction were the same. The coupling reaction conditions were: temperature 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0231] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0232] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0233] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0234] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to the solid phase support was added to 0.5 ml / μmol of 25% by mass ammonia water at 55°C for 16 hours, the solvent was removed, and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by the addition of 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride to remove the 2'-O-TBDMS protection from the ribose.

[0235] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the elution gradient was eluent 1:eluent 2 = (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0236] Detection: Purity was detected using ion exchange chromatography (IEX-HPLC); molecular weight was detected using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value ≈ the theoretical value, it indicated that the ligand was conjugated to the 3' end of the sense strand.

[0237] Step 3: Synthesis of the antisense strand

[0238] The antisense strand was synthesized using a universal solid-phase support. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification, and desalting conditions in the solid-phase synthesis of the antisense strand were the same as those in step 2 for the sense strand.

[0239] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value ≈ the theoretical value, the antisense strand was obtained.

[0240] Step 4: Synthesis of double-stranded RNAi agents

[0241] The sense chain synthesized in step 2 and the antisense chain synthesized in step 3 were mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense chain and antisense chain to form a double-stranded structure through hydrogen bonds, thereby obtaining an RNAi agent having the sense chain and antisense chain shown in Table 2.

[0242] The information of the prepared double-stranded RNAi agent is shown in the following table:

[0243] Table 2 Sequence list of double-stranded RNAi agents with L96 as ligand

[0244] In this sequence, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond; (moe) indicates that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl (i.e., 2'-O-MOE)-modified nucleotide; and L96 is N-acetylgalactosamine.

[0245] Example 2 Preparation of double-stranded RNAi agent with (CR01008) × 3 as ligand

[0246] Step 1: (CR01008)×3 ligand was prepared by the method shown in Preparation Example 2.

[0247] Step 2: Sense strand synthesis: Using phosphoramidite nucleic acid solid-phase synthesis, the three CR01008 compounds attached to a solid support were used as the initial cycle. Nucleoside monomers were then attached one by one in the 3'-5' direction according to the nucleotide sequence. Each ligation step involved deprotection, coupling, capping, and oxidation or sulfurization. The synthesis method and conditions were similar to those described in Example 1 for sense strand synthesis.

[0248] Step 3: Synthesis of antisense strand: The antisense strand was synthesized according to the method and conditions for synthesizing the antisense strand as described in Example 1.

[0249] Step 4: Synthesis of double-stranded RNAi agent: Prepared according to the method described in Example 1.

[0250] The information of the obtained double-stranded RNAi agent is shown in the following table:

[0251] Table 3 (CR01008) × 3 is the double-stranded RNAi agent sequence list of ligands

[0252] Biological detection experiments

[0253] Unless otherwise stated, the reagents and consumables (Table 4) and instruments and equipment (Table 5) used in this application were commercially available products from the following manufacturers.

[0254] Table 4 Main reagents and consumables

[0255] Table 5 Main instruments and equipment

[0256] Experiment 1 Evaluation of in vitro activity of double-stranded RNAi agents

[0257] This example uses the target gene inhibitory activity assessment method in the human liver cancer cell line HepG2 to evaluate the inhibitory activity of RZ011001 to RZ011255 on the target gene CFB in cells at the same CFB target site, with RZ000001 as a negative control;

[0258] Test sample preparation:

[0259] After centrifugation, dissolve each double-stranded RNAi agent in PBS according to the specifications of each tube to prepare a 20 μM stock solution. This stock solution was then serially diluted with PBS to a 1 μM working solution. Dose testing was performed at a final duplex concentration of 10 nM.

[0260] 96-well transfection and detection:

[0261] HepG2 cells grown to near confluence were digested with trypsin, and the cells were washed to prepare a cell suspension. 100 μL of cell suspension was added to each well of a 96-well plate, with 12,000 cells per well. The cells were cultured in a 37°C, 5% CO2 incubator. After the cells adhered for 24 hours, the DMEM medium in the 96-well plate was aspirated and 80 μL of Opti-MEM was added to each well. TM Culture medium was added, and the 96-well plate was then placed in an incubator for further incubation. 1 μL of the 1 μM working solution was dispersed in 9 μL of Opti-MEM to form the siRNA mixture. 0.3 μL of RNAiMAX was dispersed in 9.7 μL of Opti-MEM and mixed with each siRNA mixture to form a transfection complex. The transfection complex was incubated at room temperature for 10 minutes, and then the transfection complex was added to the 96-well plate at a rate of 20 μL / well. After 4 hours of incubation, 100 μL of DMEM medium supplemented with 20% FBS was added to each well, and the 96-well plate was placed in an incubator for an additional 24 hours.

[0262] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0263] Use reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 Reverse transcription primers, according to the method of reverse transcription kit specification sheet, configure 20 μ L reverse transcription system and complete reverse transcription reaction.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in HepG2 cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, primers for target gene and primers for GAPDH internal reference gene are used to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primers is shown in Table 6.

[0264] Table 6 Sequences of detection primers

[0265] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0266] Table 7 Inhibitory activity of target genes in HepG2 cells after administration of double-stranded RNAi agents

[0267] Experiment 2 Evaluation of RNAi agent activity in HDI mice

[0268] In this example, the Balb / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the same CFB target double-stranded RNAi agent with the 3' end of the sense strand conjugated to the L96 ligand on the target gene CFB, with RZ000001 as a negative control.

[0269] Plasmid construction: pcDNA-CMV-RG011 plasmid (ID: NM_001710.6) was constructed by Sangon Biotech (Shanghai) Co., Ltd.

[0270] Mouse model construction:

[0271] The Balb / c mouse hydrodynamic injection model utilizes rapid, high-pressure injection of a pcDNA-CMV-RG011 plasmid solution into the tail vein. On day 4 of the experiment, mice were injected hydrodynamically with 10 μg of pcDNA-CMV-RG011 over 5 seconds, at an injection volume of 8% of their body weight. Plasmid DNA for injection was diluted with saline, prepared immediately prior to injection, and stored at 4°C.

[0272] Animal grouping, drug administration and tissue sample collection:

[0273] 6-8 week old Balb / c mice were randomly divided into groups (all female) according to body weight, with 5 mice in each group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were dosed according to body weight, and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.1 mg / mL (calculated as a double-stranded RNAi agent) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 1 mg / kg mouse body weight (calculated as a double-stranded RNAi agent). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3). On the 4th day (recorded as D4), 5 mice of all groups were killed. The mice were subjected to gross dissection and the liver tissue of each mouse was collected. The liver tissue was cut into about 2 mm 3 Small pieces were stored with RNA later.

[0274] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0275] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo (dT) was selected. 18Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription systems and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as an internal reference gene, and the target gene and the Nero internal reference gene were detected using primers for the target gene and primers for the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 8.

[0276] Table 8 Sequences of detection primers

[0277] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table X), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The prepared reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 minutes, followed by pre-denaturation at 95°C for 20 seconds, denaturation at 95°C for 3 seconds, annealing and extension at 60°C for 30 seconds, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0278] Table 9 Inhibitory activity of target genes in Balb / c-HDI mice after administration of double-stranded RNAi agents

[0279] The results in Table 9 above show that at a dose of 1 mg / kg, RZ011281, RZ011292, RZ011293, RZ011299, RZ011301, and RZ011303 can significantly inhibit CFB mRNA expression, with an inhibitory effect of ≥80%.

[0280] Experiment 3 Evaluation of the activity of double-stranded RNAi agents in primary mouse liver cells

[0281] In this example, C57BL / 6J mouse primary liver cells were used to evaluate the inhibitory activity of the same CFB target double-stranded RNAi agent with the 3' end of the sense strand conjugated to the L96 ligand on the target gene CFB, and RZ000001 was used as a negative control;

[0282] Isolation of primary mouse liver cells: Anesthetize 6-8 week old mice, expose the abdominal cavity to find the portal vein and inferior vena cava, and wash them with preheated (37°C) HBSS-EDTA. 2+ and Mg 2+ Digestion was performed with 0.08% type IV collagenase in HBSS until the liver slowly rebounded from its elastic bulge and developed texture. The perfused liver was removed from the animal, and the cell suspension was filtered through a cell sieve to remove undigested tissue and connective tissue. The cells were then collected into a centrifuge tube. The cell suspension was centrifuged, resuspended in culture medium, and stained with trypan blue at a final concentration of 0.04% for 2 minutes to assess cell viability.

[0283] Cell plating: Dilute cells to an appropriate density with culture medium and plate at a rate of 2×10 4 The wells were inoculated in a 96-well plate, 100 μL / well.

[0284] Cell transfection: Add 1 μL of the diluted compound to a working concentration of 10 nM and gently shake the plate back and forth to mix evenly. Incubate the cell plate at 37°C in a 5% CO2 incubator for 24 hours.

[0285] The 96-well plate was removed and total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0286] Use reverse transcription kit (Thermo Fisher Scientific company, RevertAid First Strand cDNA Synthesis Kit, K1622) and select Oligo (dT) 18 reverse transcription primer, configure 20 μ L reverse transcription system according to the method of reverse transcription kit specification sheet record and complete reverse transcription reaction.Then use real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific company, TaqMan Fast Advanced Master Mix, 4444557) to detect the expression of target gene mRNA in mouse liver primary cells on fluorescence quantitative PCR instrument (Bio-Rad company CFX Opus 384).In this real-time fluorescence quantitative PCR method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as internal reference gene, use primer for target gene and primer for GAPDH internal reference gene to detect target gene and GAPDH internal reference gene respectively.The sequence of detection primer is shown in Table 10.

[0287] Table 10 Sequences of detection primers

[0288] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table X), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The prepared reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 minutes, followed by pre-denaturation at 95°C for 20 seconds, denaturation at 95°C for 3 seconds, annealing and extension at 60°C for 30 seconds, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0289] Table 11 Inhibitory activity of target genes in mouse liver primary cells after administration of double-stranded RNAi agents

[0290] The results in Table 11 and Figure 1 indicate that at a dose of 10 nM, multiple L96 vector conjugates can significantly inhibit CFB mRNA expression in mouse primary liver cells.

[0291] Experiment 4 Evaluation of RNAi agent activity in C57BL / 6J mice

[0292] This example evaluated the mRNA inhibition activity of the RNAi agent of L96 ligand in the liver tissue of C57BL / 6J mice, using RZ000001 as a negative control.

[0293] Animal grouping, drug administration and tissue sample collection:

[0294] 6-8 week old C57BL / 6J male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 5 mice per group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight, and the administration volume was 10 mL / kg of mouse body weight. Single administration was performed by subcutaneous injection in the abdomen, and each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution, i.e., the dosage of each drug conjugate was 3 mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate).

[0295] The day of administration was designated as day 0 (D0), and all mice in the groups were killed on the 7th day after administration (D7). The mice were dissected and the liver tissue of each mouse was collected. The liver tissue was cut into pieces of about 2 mm. 3 Small pieces were stored in RNA later and CFB mRNA expression levels were measured. In this real-time fluorescence quantitative PCR method, GAPDH was used as an internal reference gene. Primers targeting the target gene and the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively. See Table 10 for the sequences of the detection primers.

[0296] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0297] Table 12 Inhibitory activity of target genes in C57BL / 6J mice after administration of double-stranded RNAi agents

[0298] The results in Table 12 and Figure 2 show that at a single dose of 3 mg / kg, RZM11014, RZM11016, RZM11039, RZ011171, and RZ011255 can significantly reduce the mRNA expression level in the liver tissue of C57BL / 6J mice, with an inhibitory effect of greater than 80%.

[0299] Experiment 5 Evaluation of the in vivo activity of double-stranded RNAi agents with different ligands in C57BL / 6J mice

[0300] This example evaluates the mRNA inhibition activity of double-stranded RNAi agents with different ligands: RZ011304, RZ011305, RZ011306, RZ011307, and RZ011255 in the liver tissue of C57BL / 6J mice.

[0301] Animal grouping, drug administration and tissue sample collection:

[0302] 6-8 week old C57BL / 6J male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 5 mice per group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on body weight, and the administration volume was 10 mL / kg of mouse body weight. Single administration was performed by subcutaneous injection in the abdomen, and each drug conjugate was administered in the form of a 0.3 mg / mL (calculated as siRNA) PBS solution, i.e., the dosage of each drug conjugate was 3 mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate).

[0303] The day of administration was designated as day 0 (D0), and all mice in the groups were killed on the 7th day after administration (D7). The mice were dissected and the liver tissue of each mouse was collected. The liver tissue was cut into pieces of about 2 mm. 3 Small pieces were stored in RNA later and CFB mRNA expression levels were measured. In this real-time fluorescence quantitative PCR method, GAPDH was used as an internal reference gene. Primers targeting the target gene and the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively. See Table 10 for the sequences of the detection primers.

[0304] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0305] Table 13 Inhibitory activity of target genes in C57BL / 6J mice after administration of double-stranded RNAi agents with different ligands

[0306] The results in Table 13 and Figure 3 show that at a single dose of 3 mg / kg, RZ011304, RZ011305, RZ011306, and RZ011307 can significantly reduce the mRNA expression level in the liver tissue of C57BL / 6J mice, indicating that the activity of RZ011304, RZ011305, RZ011306, and RZ011307 is better than that of RZ011255.

[0307] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial Applicability

[0308] The present disclosure provides a complement factor B (CFB) inhibitor, comprising a double-stranded oligonucleotide, a double-stranded RNAi agent, and a pharmaceutical composition comprising the same; the present disclosure provides a method for treating, preventing, or alleviating a disease associated with dysregulation of the alternative complement pathway in a subject by administering a complement factor B (CFB) inhibitor. The present disclosure also provides a method for inhibiting CFB expression by administering a CFB-specific inhibitor (double-stranded RNAi agent) to a subject.

Claims

1. A double-stranded oligonucleotide (dsRNA) for inhibiting complement factor B (CFB) gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides of any sequence shown in SEQ ID NO:1-SEQ ID NO:255 in Table 1 or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; and / or the antisense strand comprising at least 15 consecutive nucleotides of any sequence shown in SEQ ID NO:256-SEQ ID NO:510 in Table 1 or a nucleotide sequence that differs from the at least 15 consecutive nucleotides by no more than 3 nucleotides; Preferably, the sense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1, and / or the antisense strand comprises a nucleotide sequence that differs by no more than 3 nucleotides from any of the sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1; in, The antisense strand is complementary or substantially complementary to the sense strand; the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides.

2. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1 by no more than 1 or 2 nucleotides, and / or the antisense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1 by no more than 1 or 2 nucleotides; Preferably, the sense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 255 in Table 1 by no more than 1 nucleotide, and / or the antisense strand comprises a nucleotide sequence that differs from any of the sequences shown in SEQ ID NO: 256 to SEQ ID NO: 510 in Table 1 by no more than 1 nucleotide; Further preferably, the sense strand comprises any one of the nucleotide sequences shown in SEQ ID NO:1-SEQ ID NO:255 in Table 1, and / or the antisense strand comprises any one of the nucleotide sequences shown in SEQ ID NO:256-SEQ ID NO:510 in Table 1.

3. The double-stranded oligonucleotide according to any one of claims 1 to 2, characterized in that The sense strand or the antisense strand comprises any sense strand or antisense strand selected from the duplexes shown in the following numbers: RN011001, RN011005, RN011006, RN011007, RN011018, RN011021, RN011022, RN011033, RN011034, RN011035, RN011038, RN011052, RN011076, RN011097, RN011098, RN011101, RN011102, RN011103, RN011104, RN011106, RN011107, RN011109 , RN011113, RN011114, RN011117, RN011123, RN011124, RN011125, RN011140, RN01115, RN011151, RN011152, RN011156, RN011160, RN011171, RN011183, RN011193, RN011194, RN011199, RN011201, RN011202, RN011233, RN011238, RN011241, RN011242, RN011243, RN011254, RN011255; Preferably, the sense strand or the antisense strand comprises a nucleotide sequence selected from the duplex shown in No. RN011255.

4. The double-stranded oligonucleotide according to any one of claims 1 to 2, characterized in that The double-stranded oligonucleotide comprises one or more selected from the duplex group shown in the following numbers: RN011001, RN011005, RN011006, RN011007, RN011018, RN011021, RN011022, RN011033, RN011034, RN011035, RN011038, RN011052, RN011076, RN011097, RN011098, RN011101, RN011102, RN011103, RN011104, RN011106, RN011107, RN011109 011113、RN011114、RN011117、RN011123、RN011124、RN011125、RN011140、RN01115、RN011151、RN011152、RN011156、RN011160、RN011171 N011183, RN011193, RN011194, RN011199, RN011201, RN011202, RN011233, RN011238, RN011241, RN011242, RN011243, RN011254, RN011255; Preferably, the double-stranded oligonucleotide comprises the duplex nucleotide sequence shown in No. RN011255.

5. The double-stranded oligonucleotide according to any one of claims 1 to 4, characterized in that All nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides; Optionally, the antisense strand and the sense strand have a length of 17-25 nucleotides; Optionally, at least one of the antisense strand and the sense strand comprises a 3'-terminal overhang of 1-2 nucleotides.

6. The double-stranded oligonucleotide of claim 5, wherein the modified nucleotide is selected from the group consisting of 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more of the nucleotides substituted by OR; wherein, n is 1 or 2, R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, amino; Preferably, the modified nucleotide is selected from 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O(CH2) n One or more of the nucleotides substituted by OR; Further preferably, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 2 or Table 3, and / or, the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 2 or Table 3.

7. A double-stranded RNAi agent for inhibiting the expression of complement factor B (CFB) gene, characterized in that: The double-stranded RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are complementary or substantially complementary to form a double-stranded region, wherein the substantially complementary means that the mismatch between the sense strand and the antisense strand in the double-stranded region does not exceed 3 nucleotides, and the double-stranded region is represented by the following formula (I): SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (I), Wherein, SS represents the sense strand, and AS represents the antisense strand; All nucleotides of the sense strand and the antisense strand are modified nucleotides; The Ns each independently represent the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides; The Xs are each independently a 2'-methoxyethyl modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-O(CH2) n OR substituted modified nucleotides; wherein n is 1 or 2, and R is selected from: substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy; wherein the substituent is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, hydroxyl, and amino; Said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from an integer of 0-3; b' is selected from an integer of 4-13; q' is selected from an integer of 0-4; c' is selected from an integer of 3-9; r' is selected from an integer of 0-3; d' is selected from an integer of 0-9; a is selected from an integer of 4-7; p is selected from an integer of 0-1; b is selected from an integer of 4-8; q is selected from an integer of 0-4; c is selected from an integer of 6-10; Furthermore, p', q', r', p, and q are not all 0 at the same time, and 0≤q'+r'≤4.

8. The double-stranded RNAi agent according to claim 7, characterized in that a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; wherein the N is selected from 2'-O-methyl modified nucleotides and 2'-fluoro-modified nucleotides, and the nucleotide X is a 2'-O-methoxyethyl modified nucleotide; Preferably, the duplex includes at least one 2'-O-methoxyethyl modification or 2'-O(CH2) n OR-modified nucleotides; Further preferably, the double-stranded RNAi agent comprises a duplex consisting of a sense strand and an antisense strand modified as follows, in the direction from the 5' end to the 3' end: The just chain is: GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmAm, or GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmsAm; The antisense strand is: UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfAmAfUmCmCmsUmsCm, or UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfAmAfUmCmCmsUmsCm, or UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfA(moe)AfUmCmCmsUmsCm, or UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfA(moe)AfUmCmCmsUmsCm, or UmsUfsAmUmAmGfAmAmAfAmsCmCmCmAfA(moe)AfUmCmCmsUmsCm.

9. The double-stranded RNAi agent according to any one of claims 7 to 8, characterized in that The nucleotides of the double-stranded RNAi sense strand and / or antisense strand are all modified nucleotides; Wherein, in the direction from the 5' end to the 3' end, at least three of the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least four of the nucleotides at positions 2, 6, 9-12, 14 and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides; Optionally, the sense strand and / or the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

10. The double-stranded RNAi agent according to claim 9, characterized in that In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides; Furthermore, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and any nucleotide selected from positions 9, 10, 11, or 12 is a 2'-fluoro-modified nucleotide, and the nucleotides at the remaining positions are 2'-O-methyl-modified or 2'-O-methoxyethyl-modified nucleotides.

11. The double-stranded RNAi agent according to claim 9 or 10, characterized in that In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; Furthermore, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluoro-modified nucleotides, and: any nucleotide selected from positions 9, 10, 11, or 12 is a 2'-fluoro-modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl-modified nucleotide, and the remaining positions are 2'-O-methyl-modified nucleotides.

12. The double-stranded RNAi agent according to any one of claims 7 to 8, characterized in that The nucleotides of the sense strand and / or the antisense strand are all modified nucleotides; and the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(4): (1) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; (2) From the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated nucleotides, and the remaining positions are 2'-O-methyl modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 9, 14, and 16 in the nucleotide sequence of the antisense strand are 2'-fluorinated modified nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 12, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated nucleotides, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides.

13. The double-stranded RNAi agent according to any one of claims 7 to 12, characterized in that The double-stranded RNAi agent further comprises a ligand; Optionally, the ligand is conjugated to the 3' end of the sense strand of the double-stranded RNAi agent; Optionally, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached via a linker.

14. The double-stranded RNAi agent according to claim 13, characterized in that The ligands are as follows: Wherein, * represents the attachment site of the oligonucleotide molecule; m is 1, 2, 3 or 4; Z, p, q, R3, L and Y are defined as follows: Z is independently selected from hydroxyl or thiol; p is independently selected from 1, 2 or 3; q is independently selected from 1, 2 or 3; each R3 is independently selected from H, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; L is a C1-C20 alkylene chain, or one or more C atoms in the chain are substituted by any substituent selected from O, S, NH or -NH-C(O)-; Y is each independently selected from NH, O or S.

15. The double-stranded RNAi agent according to claim 13 or 14, characterized in that The ligand structure is as follows:

16. The double-stranded RNAi agent according to any one of claims 7 to 15, characterized in that As shown below: Wherein, Nu represents a double-stranded oligonucleotide molecule; Preferably, the sense strand of the double-stranded oligonucleotide comprises any modified sense strand nucleotide sequence shown in Table 3, and / or, the antisense strand comprises any modified antisense strand nucleotide sequence shown in Table 3; Optionally, the ligand is attached to the 3' end of the sense strand.

17. The double-stranded RNAi agent according to claim 12, characterized in that The 3' end of the sense strand in the double-stranded RNAi agent is covalently coupled to a ligand with the following structure:

18. The double-stranded RNAi agent according to claim 17, characterized in that The double-stranded RNAi agent is selected from the group consisting of duplexes numbered RZ011304, RZ011305, RZ011306, and RZ011307; Preferably, the sense strand in the double-stranded RNAi agent is: 5'-GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmAm_(CR01008×3)-3', or 5'-GmsGmsAmUmUmUmGfGfGfUfUmUmUmCmUmAmUmAmsAm_(CR01008×3)-3'; And the antisense strand is: 5'-UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfAmAfUmCmCmsUmsCm-3', or 5'-UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfAmAfUmCmCmsUmsCm-3', or 5'-UmsUfsAmUmAmGfAmAmAfAmCmCmCmAfA(moe)AfUmCmCmsUmsCm-3', or 5'-UmsUfsAmUmAmGfAmAmAmAmCmCfCmAfA(moe)AfUmCmCmsUmsCm-3', or 5'-UmsUfsAmUmAmGfAmAmAfAmsCmCmCmAfA(moe)AfUmCmCmsUmsCm-3'; wherein Am, Cm, Gm and Um are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine phosphate and 2'-O-methyluridine phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; A(moe) is 2'-O-methoxyethyladenosine-3'-phosphate, and s is phosphorothioate; Among them, CR01008×3 is a ligand with the following structure:

19. The double-stranded RNAi agent according to any one of claims 7 to 18, characterized in that: The sense strand and / or the antisense strand further comprises a 3' and / or 5' extension or overhang of 1-3 nucleotides in length; Optionally, the sense strand is 17-21 nucleotides long, and the antisense strand is 19-23 nucleotides long; Optionally, the sense strand and / or the antisense strand of the RNAi agent independently comprises one or more phosphorothioate internucleoside linkages; Optionally, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends, or the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' and 5' ends; Optionally, the double-stranded region is 19 to 23 nucleotide pairs in length; or, the double-stranded region is 19 to 21 nucleotide pairs in length; Optionally, at least one strand of the double-stranded region of the RNAi agent comprises a 3' overhang of 1-2 nucleotides.

20. The double-stranded RNAi agent of claim 7, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein each strand is 14 to 25 nucleotides in length, and the antisense strand comprises a region complementary to a portion of an mRNA encoding CFB, wherein the double-stranded region is represented by the following Formula II: SS: 5'-(N)a'-(X)p'-(N)b'-(X)q'-(N)c'-(X)r'-(N)d'-3' AS: 3'-(N)a-(X)p-(N)b-(X)q-(N)c-5' (II) in, SS represents the sense strand, AS represents the antisense strand; the Ns each independently represent the following modified nucleotides: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides; the Xs each independently represent 2'-O-methoxyethyl modified nucleotides; wherein said a, a', p, p', b, b', q, q', c, c', r', d' each independently represents the number of nucleotides, wherein: a' is selected from an integer of 3-8; p' is selected from 0 or 1; b' is selected from an integer of 4-13; q' is selected from 0 or 1; c' is selected from an integer of 3-9; r' is selected from 0 or 1; d' is selected from an integer of 1-8; a is selected from an integer of 4-7; p is selected from 1; b is selected from an integer of 4-8; q is selected from 0 or 1; c is selected from an integer of 6-10; and p', q', r', p, q are not all 0 at the same time, 0≤q'+r'≤2; Furthermore, (N)a contains at least one fluorinated modified nucleotide, and the 16th position of the antisense strand starting from the 5'-end is a fluorinated modified nucleotide; (N)b contains at least one fluorinated modified nucleotide, and the 14th position of the antisense strand starting from the 5'-end is a fluorinated modified nucleotide; (N)c contains at least two fluorinated modified nucleotides. nucleotides, and starting from the 5'-end, the 2nd and 6th positions of the antisense strand are both fluorinated modified nucleotides; starting from the 5'-end, the first 4 nucleotides of (N)b' contain at least 2 fluorinated modified nucleotides; Preferably, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are fluorinated modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl modified nucleotides; and, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense strand are 2'-fluorinated modified nucleotides, and: any nucleotide selected from positions 9 or 10 or 11 or 12 is a 2'-fluorinated modified nucleotide, the nucleotide at position 15 is a 2'-O-methoxyethyl modified nucleotide, and the remaining positions are 2'-O-methyl modified nucleotides; Wherein, the sense chain is conjugated to a ligand.

21. The double-stranded RNAi agent of claim 20, wherein the ligand structure is as follows:

22. Use of the double-stranded oligonucleotide according to any one of claims 1 to 6 and the double-stranded RNAi agent according to any one of claims 7 to 21 in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by a complement factor B gene; Optionally, the disease or condition comprises renal disease, systemic lupus erythematosus and related diseases, macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria, and rheumatoid arthritis; Preferably, the renal disease includes C3 glomerulopathy, lupus nephritis, IgA nephropathy, diabetic nephropathy, membranous nephropathy and polycystic kidney disease.

23. A pharmaceutical composition, characterized in that The invention comprises the double-stranded oligonucleotide according to any one of claims 1 to 6 and the double-stranded RNAi agent according to any one of claims 7 to 21, and further comprises pharmaceutically optional auxiliary materials.

24. A kit, characterized in that The kit comprises the double-stranded oligonucleotide according to any one of claims 1 to 6 and the double-stranded RNAi agent according to any one of claims 7 to 21 or a pharmaceutical composition thereof.

25. A method for inhibiting complement factor B gene expression, characterized in that: The method comprises administering to a subject a double-stranded oligonucleotide according to any one of claims 1 to 6, or a double-stranded RNAi agent according to any one of claims 7 to 21, or a pharmaceutical composition according to claim 23; Optionally, the expression of complement factor B is inhibited by at least 50%, 60%, 70%, 80%, 90% or 95%; Optionally, wherein inhibiting the expression of complement factor B reduces the level of complement factor B protein in the serum of the subject by at least 50%, 60%, 70%, 80%, 90% or 95%.

26. A method for alleviating, treating and / or preventing a disease or condition mediated by complement factor B, characterized in that: The method comprises administering to a subject a double-stranded oligonucleotide according to any one of claims 1 to 6, or a double-stranded RNAi agent according to any one of claims 7 to 21, or a pharmaceutical composition according to claim 23; Optionally, the complement factor B-mediated disease or condition comprises a disease associated with the level of mRNA expressed by the complement factor B gene; Optionally, the disease or condition comprises renal disease, systemic lupus erythematosus and related diseases, macular degeneration, atypical hemolytic uremic syndrome, thrombotic microangiopathy, myasthenia gravis, ischemia and reperfusion injury, paroxysmal nocturnal hemoglobinuria and rheumatoid arthritis Preferably, the renal disease includes C3 glomerulopathy, lupus nephritis, IgA nephropathy, diabetic nephropathy, membranous nephropathy and polycystic kidney disease.

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