Sirna for inhibiting CFB gene expression, and modifier and use thereof

Abstract

Provided is a CFB-targeting double-stranded RNA molecule, specifically an siRNA. The provided siRNA can effectively inhibit the CFB expression, and can be used for preventing and treating diseases caused by up-regulated CFB expression or diseases associated with abnormal activation of an alternative complement pathway.

Description

A siRNA for inhibiting CFB gene expression and its modifications and applications

[0001] Cross-references to related applications

[0002] This patent application claims priority to Chinese Patent Application No. CN2024110435145, filed on July 31, 2024, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention belongs to the field of biomedicine and relates to an siRNA for inhibiting CFB gene expression, its modifications, and applications. Background Technology

[0004] The intrinsic components of the complement system typically exist in an inactive form, activating through a cascade of enzymatic reactions. This mechanism primarily involves three pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). Despite their different mechanisms, activation of all three pathways ultimately leads to the formation of C3 convertase. C3 convertase cleaves the complement component C3, and the resulting C3b fragment subsequently participates in the formation of C5 convertase. C5 convertase cleaves C5, triggering subsequent reactions that ultimately form the membrane attack complex (MAC). MAC plays a crucial physiological and pathological role through its cytolytic effect.

[0005] The alternative pathway, also known as the bypass pathway, is one of the three pathways mentioned above. It is antibody-independent and activated by certain bacteria, fungi, bacterial lipopolysaccharides (endotoxins), yeast polysaccharides, dextran, and aggregated IgA and IgG4. These activators can bind to C3b or hydrated C3. With the participation of key factors including complement factor B (CFB), factor D, and factor P, C3 convertase and C5 convertase are formed successively, ultimately leading to the membrane attack complex (MAC). CFB, primarily synthesized by the liver and macrophages, is an indispensable core component for the activation of the alternative pathway.

[0006] Abnormal or excessive activation of alternative pathways, also known as bypass pathways, is closely associated with a variety of diseases. These include neurological disorders such as Alzheimer's disease (AD), neuromyelitis optica (NMO), and myasthenia gravis (gMG); eye diseases such as age-related macular degeneration, uveitis, and glaucoma; kidney diseases such as atypical hemolytic uremic syndrome, C3 glomerulonephritis, and IgA nephropathy; and blood disorders such as cold agglutinin disorders, paroxysmal nocturnal hemoglobinuria, and thrombotic microangiopathy. Given that caffeine-rich protein (CFB) is a key factor in bypass pathways, it is believed that inhibiting CFB activation can effectively suppress, alleviate, or treat the aforementioned diseases.

[0007] Currently, the only approved drug targeting CFB is Novartis' small molecule chemical drug, iprocoman hydrochloride. Compared with traditional small molecule drugs, siRNA drugs have advantages such as high efficacy, high stability, low immunogenicity, and low risk of side effects. Therefore, developing siRNA drugs targeting CFB based on siRNA technology will have significant clinical therapeutic value and a promising market prospect. Summary of the Invention

[0008] The purpose of this invention is to provide a highly efficient siRNA for inhibiting CFB expression.

[0009] In a first aspect, the present invention provides an siRNA comprising at least partially paired sense and antisense strands forming a double-stranded region, wherein the length of the double-stranded region is 15-30, 23-27, 21-23, 19-21, 17-25, 17-23, or 17-19 base pairs.

[0010] Preferably, in the siRNA described above, the sense strand and the antisense strand may each have 15-30 nucleotides.

[0011] More preferably, the sense strand of the siRNA comprises the following nucleotide sequence:

[0012] A1) The nucleotide sequence represented by any odd-numbered sequence from SEQ ID NO.1 to SEQ ID NO.394; or

[0013] A2) A nucleotide sequence that is more than 90% homologous to any of the odd-numbered nucleotide sequences in SEQ ID NO.1 to SEQ ID NO.394.

[0014] More preferably, the antisense strand of the siRNA comprises the following nucleotide sequence:

[0015] A3) The nucleotide sequence represented by any even number in SEQ ID NO.1 to SEQ ID NO.394; or

[0016] A4) A nucleotide sequence that is more than 90% homologous to any even-numbered nucleotide sequence in SSEQ ID NO.1 to SEQ ID NO.394.

[0017] According to a specific embodiment of the present invention, the siRNA provided by the present invention comprises one sense strand and one antisense strand selected from the following:

[0018] The sense strand comprises the nucleotide sequence represented by any odd number n from SEQ ID NO.1 to SEQ ID NO.394, and the antisense strand comprises the nucleotide sequence represented by any even number n+1 from SEQ ID NO.1 to SEQ ID NO.394, where n is an odd number between 1 and 394.

[0019] Furthermore, at least one nucleotide in the sense or antisense strand of the siRNA provided by the present invention can be a modified nucleotide.

[0020] The modified nucleotide may be selected from at least one of the following: 2'-methoxy-modified nucleotides, 2'-fluorine-modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, and nucleotides containing 5'-phosphate ester mimics.

[0021] Furthermore, the siRNA provided by the present invention can also be modified with 5'-(E)-VP at the 5' end of the antisense strand (that is, the 5' hydroxyl group of the first nucleotide at the 5' end of the antisense strand is replaced with (E)-ethylene phosphate).

[0022] Furthermore, the siRNA provided by this invention may also have an inverse abase (invab) attached to the 5' end and / or 3' end of the positive strand.

[0023] In some implementations, when invab is attached to the 5' end of the justice chain, the structure is as follows: It can also be modified with thiophosphates (invab): When invab is connected at the 3' end of the justice chain, it is as follows: When L96 is coupled at the 3' end, the following formula is shown:

[0024] According to specific embodiments of the present invention, the present invention provides modified siRNAs, which are shown in Tables 18 and 19 below. Specifically, the siRNAs comprise a sense strand and an antisense strand as shown below:

[0025] (1) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.430;

[0026] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0027] Antonym chain: VPAmsGfsUmCmAmUmAmAmAfAmUmUfCmAfGmGfAmAmUmsUmsCm (SEQ ID NO.430);

[0028] (2) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.412;

[0029] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0030] Antonym chain: AmsGfsUmCmAmUmAmAmAfAmUmUfCmAfGmGfAmAmUmsUmsCm(SEQ ID NO.412);

[0031] (3) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.401;

[0032] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0033] Antonym chain: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm(SEQ ID NO.401);

[0034] (4) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.402;

[0035] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0036] Antonym chain: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCfUmCfAmCmAmsUmsUm(SEQ ID NO.402);

[0037] (5) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.403;

[0038] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0039] Antonym chain: AmsGfsAmGmAmUfCmUmCmAfUmC(d)AmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.403);

[0040] (6) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.404;

[0041] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0042] Antonym chain: AmsGfsAmGmAmUfCmUmCmAfUmC(d)AmCfUmCfAmCmAmsUmsUm(SEQ ID NO.404);

[0043] (7) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.405;

[0044] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0045] Antonym chain: AmsGfsAmGmAmUmCmUfCmAfUmC(d)AmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.405);

[0046] (8) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.406;

[0047] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0048] Antonym chain: AmsGfsAmGfAmUmCmUmCmAmUmCfAmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.406);

[0049] (9) The sense strand contains the nucleotide sequence shown in SEQ ID NO.407, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.408;

[0050] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGmAfUmGmAmGmAmUmCmUmCmUmUms(Invab)(SEQ ID NO.407);

[0051] Antonym chain: AmsGfsAmGmAmUmCmUmCmAmUmC(d)AmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.408);

[0052] (10) The sense strand contains the nucleotide sequence shown in SEQ ID NO.397, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.398;

[0053] Chain of Justice: AmsUmsUmCmCmUmGfAfAfUmUmUmUmAmUmGmAmCmUm(SEQ ID NO.397);

[0054] Antonyms: AmsGfsUmCmAmUfAmAmAmAmUmUmCmAfGmGfAmAmUmsUmsCm(SEQ ID NO.398);

[0055] (11) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.410;

[0056] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0057] Antonym chain: AmsGfsUmCmAmUmAmAfAm(Im)UmUfCmAfGmGfAmAmUmsUmsCm(SEQ ID NO.410);

[0058] (12) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.411;

[0059] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0060] Antonym chain: AmsGfsUmCmAmUmAmAmAfAmUmUfCmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.411);

[0061] (13) The sense strand contains the nucleotide sequence shown in SEQ ID NO.395, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.396;

[0062] Chain of Justice: UmsGmsUmGmAmGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUm(SEQ ID NO.395);

[0063] Antonym chain: AmsGfsAmGmAmUfCmUmCmAmUmCmAmCfUmCfAmCmAmsUmsUm(SEQ ID NO.396);

[0064] (14) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.413;

[0065] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0066] Antonym chain: AmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.413);

[0067] (15) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.414;

[0068] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0069] Antonym chain: AmsGfsUmCmAmUfAmAmAmA(d)UmUfCmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.414);

[0070] (16) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.415;

[0071] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0072] Antonym chain: AmsGfsUmCmAmUfAmAmAmAfUmT(d)CmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.415);

[0073] (17) The sense strand contains the nucleotide sequence shown in SEQ ID NO.416, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.417;

[0074] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.416);

[0075] Antonym chain: AmsGfsUmCmAmUmAmAmAmAmUmT(d)CmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.417);

[0076] (18) The sense strand contains the nucleotide sequence shown in SEQ ID NO.395, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.418;

[0077] Chain of Justice: UmsGmsUmGmAmGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUm(SEQ ID NO.395);

[0078] Antonym chain: VPAmsGfsAmGmAmUfCmUmCmAmUmCmAmCfUmCfAmCmAmsUmsUm(SEQ ID NO.418);

[0079] (19) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.419;

[0080] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0081] Antonym chain: VPAmsGfsAmGmAmUmCmUmCfAmUmCfAmCf(Im)CfAmCmAmsUmsUm(SEQ ID NO.419);

[0082] (20) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.420;

[0083] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0084] Antonym chain: VPAmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm(SEQ ID NO.420);

[0085] (21) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.421;

[0086] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0087] Antonym chain: VPAmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCfUmCfAmCmAmsUmsUm(SEQ ID NO.421);

[0088] (22) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.422;

[0089] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0090] Antonym chain: VPAmsGfsAmGmAmUfCmUmCmAfUmC(d)AmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.422);

[0091] (23) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.423;

[0092] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0093] Antonym chain: VPAmsGfsAmGmAmUfCmUmCmAfUmC(d)AmCfUmCfAmCmAmsUmsUm(SEQ ID NO.423);

[0094] (24) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.424;

[0095] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0096] Antonym chain: VPAmsGfsAmGmAmUmCmUfCmAfUmC(d)AmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.424);

[0097] (25) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.425;

[0098] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0099] Antonym chain: VPAmsGfsAmGfAmUmCmUmCmAmUmCfAmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.425);

[0100] (26) The sense strand contains the nucleotide sequence shown in SEQ ID NO.407, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.426;

[0101] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGmAfUmGmAmGmAmUmCmUmCmUmUms(Invab)(SEQ ID NO.407);

[0102] Antonym chain: AmsGfsAmGmAmUmCmUmCmAmUmC(d)AmCfUmCf(Im)CmAmsUmsUm(SEQ ID NO.426);

[0103] (27) The sense strand contains the nucleotide sequence shown in SEQ ID NO.397, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.427;

[0104] Chain of Justice: AmsUmsUmCmCmUmGfAfAfUmUmUmUmAmUmGmAmCmUm(SEQ ID NO.397);

[0105] Antonym chain: VPAmsGfsUmCmAmUfAmAmAmAmUmUmCmAfGmGfAmAmUmsUmsCm(SEQ ID NO.427);

[0106] (28) The sense strand comprises the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.428;

[0107] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0108] Antonym chain: VPAmsGfsUmCmAmUmAmAfAm(Im)UmUfCmAfGmGfAmAmUmsUmsCm(SEQ ID NO.428);

[0109] (29) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.429;

[0110] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0111] Antonym chain: VPAmsGfsUmCmAmUmAmAmAfAmUmUfCmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.429);

[0112] (30) The sense strand contains the nucleotide sequence shown in SEQ ID NO.399, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.400;

[0113] Chain of Justice: (Invab)sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUmCmUms(Invab)(SEQ ID NO.399);

[0114] Antonym chain: AmsGfsAmGmAmUmCmUmCfAmUmCfAmCf(Im)CfAmCmAmsUmsUm(SEQ ID NO.400);

[0115] (31) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.431;

[0116] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0117] Antonym chain: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.431);

[0118] (32) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.432;

[0119] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0120] Antisense strand: VPAmsGfsUmCmAmUfAmAmAmA(d)UmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 432);

[0121] (33) The sense strand contains the nucleotide sequence shown in SEQ ID NO.409, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.433;

[0122] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.409);

[0123] Antonym chain: VPAmsGfsUmCmAmUfAmAmAmAfUmT(d)CmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.433);

[0124] (34) The sense strand contains the nucleotide sequence shown in SEQ ID NO.416, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.434;

[0125] Chain of Justice: (Invab)sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms(Invab)(SEQ ID NO.416);

[0126] Antonym chain: VPAmsGfsUmCmAmUmAmAmAmAmUmT(d)CmAf(Im)GfAmAmUmsUmsCm(SEQ ID NO.434).

[0127] In the above nucleotide sequence, m indicates that the nucleotide to the left of m is a 2'-methoxy modified nucleotide; s indicates thiophosphate modification, that is, the two nucleotides adjacent to the letter s are linked by thiophosphate groups; f indicates that the nucleotide to the left of f is a 2'-fluoro modified nucleotide; (d) indicates that the nucleotide to the left of (d) is a 2'-deoxy modified nucleotide; (invab) indicates the above-mentioned inverse abase-free nucleotide; VP indicates that the nucleotide to the right of VP is a 5'-(E)-vinyl phosphate modified nucleotide.

[0128] Furthermore, the siRNA provided by the present invention has a ligand attached to the 3'-end or 5'-end of the positive strand or to any nucleotide in the positive strand. For example, the siRNA shown in items (1) to (34) above has a ligand attached to the 3'-end or 5'-end of the positive strand or to any nucleotide in the positive strand.

[0129] In one embodiment, the 3' end of the positive strand of the siRNA described in items (1) to (34) above is attached to a ligand.

[0130] Preferably, the ligand is a ligand containing N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0131] Furthermore, in the siRNA provided by the present invention, the ligand can be linked to the siRNA via a linker.

[0132] Furthermore, the ligand is L96 as shown in the following formula:

[0133] According to a specific embodiment of the present invention, the siRNA and ligand connection method provided by the present invention is as follows: L96 is coupled to the 3' end of the positive strand of the modified siRNA.

[0134] According to a specific embodiment of the present invention, the siRNA conjugate formed by L96 and siRNA molecules has the following structure:

[0135] Furthermore, the siRNA is selected from those shown in Table 20 below.

[0136] In a second aspect, the present invention provides a composition comprising the siRNA described in the first aspect of the present invention.

[0137] The compositions provided by this invention may also include pharmaceutically acceptable carriers.

[0138] Furthermore, the pharmaceutical composition may also contain an unbuffered solution. This unbuffered solution may be physiological saline or water. Alternatively, the pharmaceutical composition may also contain a buffer solution. This buffer solution contains acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof. For example, the buffer solution is phosphate-buffered saline (PBS).

[0139] The pharmaceutically acceptable carrier may be an excipient, stabilizer, suspending agent, or diluent, as is well known to those skilled in the art.

[0140] Thirdly, the present invention provides a cell comprising the siRNA described in the first aspect of the present invention.

[0141] Fourthly, the present invention provides the use of the siRNA described in the first aspect, the composition described in the second aspect, or the cell described in the third aspect in the preparation of a medicament for treating diseases caused by abnormal CFB expression.

[0142] Furthermore, the disease caused by abnormal CFB expression can be a disease caused by upregulation of CFB expression.

[0143] Furthermore, the diseases caused by abnormal CFB expression include diseases related to abnormal activation of the complement bypass pathway.

[0144] Fifthly, the present invention provides a method for treating diseases caused by abnormal CFB expression, the method comprising administering to a subject in need the siRNA described in the first aspect, the composition described in the second aspect, or the cells described in the third aspect.

[0145] Furthermore, the disease caused by abnormal CFB expression can be a disease caused by upregulation of CFB expression.

[0146] Furthermore, the diseases caused by abnormal CFB expression include diseases related to abnormal activation of the complement bypass pathway.

[0147] The method for treating diseases caused by abnormal CFB expression provided by this invention can be used for non-diagnostic or non-therapeutic purposes, or for scientific research purposes.

[0148] Sixthly, the present invention provides a method for inhibiting CFB expression in cells, the method comprising:

[0149] (a) Contacting the cells with the siRNA described in the first aspect, the composition described in the second aspect, or the cells described in the third aspect.

[0150] (b) Maintain the cells produced in step (a) for a period of time sufficient for the degradation of the CFB gene mRNA transcript to suppress CFB expression in the cells.

[0151] Further, in step (a), the cells may be located within the body of the subject, who may suffer from a disease caused by abnormal CFB expression. The subject may be an animal, such as a mammal, including primates (e.g., humans or non-human primates, such as monkeys or chimpanzees), or non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, or whales). In some embodiments, the subject is a mouse.

[0152] Further, in step (b), the inhibition of CFB expression in cells means that the CFB expression level is reduced by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% compared to before contact.

[0153] Furthermore, the disease caused by abnormal CFB expression can be a disease caused by upregulation of CFB expression.

[0154] Furthermore, the diseases caused by abnormal CFB expression include diseases related to abnormal activation of the complement bypass pathway.

[0155] The method for inhibiting CFB expression in cells provided by this invention can be used for non-diagnostic or non-therapeutic purposes, or for scientific research purposes.

[0156] Furthermore, the diseases caused by abnormal CFB expression as described in the fourth, fifth, or sixth aspect of this invention include, but are not limited to, neurological diseases such as Alzheimer's disease (AD), autoimmune neurological diseases, neuromyelitis optica (NMO), and myasthenia gravis (gMG); eye diseases such as age-related macular degeneration (AMD), age-related macular atrophy, uveitis, and glaucoma; kidney diseases (such as atypical hemolytic uremic syndrome, C3 glomerulonephropathy, IgA nephropathy, membranoproliferative glomerulonephritis, and lupus nephritis); and blood diseases. Cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, thrombotic microangiopathy, septic shock, diabetic retinopathy, retinitis pigmentosa, macular edema, Behçet's uveitis, multifocal choroiditis, Fuku-Koyanagi-Harada syndrome, intermediate uveitis, shotgun retinochoroiditis, sympathetic ophthalmia, ocular cicatricial pemphigoid, ocular pemphigoid, non-arteritis-ischemic optic neuropathy, postoperative inflammation, retinal vein occlusion, neurological disorders, multiple sclerosis, stroke, Gibbs-Barré syndrome, traumatic brain injury, Parkinson's disease, discomfort Suitable or undesirable complement activation disorders, hemodialysis complications, hyperacute allogeneic graft rejection, xenograft rejection, interleukin-2 induced toxicity during IL-2 therapy, inflammation in autoimmune diseases, Crohn's disease, adult-onset respiratory distress syndrome, myocarditis, ischemia-reperfusion syndrome, myocardial infarction, balloon angioplasty, post-pump syndrome during cardiopulmonary shunt or renal shunt surgery, atherosclerosis, hemodialysis, renal ischemia, aortic reconstruction, mesenteric artery reperfusion after infectious diseases or sepsis, immune complex disorders, and autoimmune diseases. Diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, liver fibrosis, hemolytic anemia, myasthenia gravis, tissue regeneration, nerve regeneration, dyspnea, hemoptysis, ARDS, asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and infarction, fibrotic dust disease, pulmonary fibrosis, asthma, allergic reactions, bronchoconstriction, allergic pneumonia, parasitic diseases, Goodman's syndrome, pulmonary vasculitis, oligoimmune vasculitis, immune complex-related inflammation, antiphospholipid syndrome, glomerulonephritis, and obesity, etc.

[0157] In a seventh aspect, the present invention provides the use of the siRNA described in the first aspect, the composition described in the second aspect, or the cell described in the third aspect in the preparation of a medicament for inhibiting CFB expression in cells.

[0158] The inhibition of cell expression means that the siRNA or the drug prepared from the siRNA can reduce the CFB expression level by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0159] In this invention, "modified nucleotides" include nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosyl group with another group, or nucleotides or nucleotide analogs formed by replacing the 5'-hydroxyl group of the ribosyl group with another group, or nucleotides in which the bases on the nucleotide are modified bases. "Methoxy-modified nucleotides" refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group. "Fluoro-modified nucleotides" refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0160] In one embodiment of the present invention, the modified nucleotides may be linked by thiophosphate groups.

[0161] In one embodiment of the present invention, a "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, having the structure shown in formula (1). The non-fluorinated nucleotide is independently selected from nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group.

[0162] In one embodiment of the present invention, the nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group is well known to those skilled in the art, and these nucleotides may be selected from one of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0163] In one embodiment of the present invention, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2), i.e., methoxy modified; the 2'-substituted alkoxy modified nucleotide, for example, can be a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3); the 2'-amino (2'-NH2) modified nucleotide is shown in formula (4); and the 2'-deoxynucleotide (DNA) is shown in formula (5), wherein Base represents a base such as A, U, G, T, C, I.

[0164] In one embodiment of the present invention, the nucleotide analogue refers to a group that can replace a nucleotide in nucleic acids, but whose structure is different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide.

[0165] In one embodiment of the present invention, the nucleotide analog may be a heteronucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0166] In one embodiment of the invention, the bridged nucleic acid (BNA) refers to a restricted or inaccessible nucleotide. The BNA may contain a bridging structure with a "fixed" C3'-endoglucan condensation of a five-membered ring, a six-membered ring, or a seven-membered ring. Typically, the bridge is incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide.

[0167] In one embodiment of the present invention, the BNA may be LNA, ENA, cET BNA, etc., wherein LNA is as shown in formula (6), ENA is as shown in formula (7), and cET BNA is as shown in formula (8):

[0168] In one embodiment of the present invention, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups.

[0169] In one embodiment of the present invention, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphate diester bond of the phosphate ester group with a sulfur atom.

[0170] In one embodiment of the invention, the phosphate group having the modifying group is a thiophosphate group having the structure shown in formula (9). In one embodiment of the invention, the nucleotide linked to the thiophosphate group is shown in formula (10), and the thiophosphate group linkage is present at least at one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof.

[0171] In one embodiment of the present invention, the VP-modified nucleotide is a vinyl phosphate ester modified. In one embodiment of the present invention, the VP-modified and methoxy-modified nucleotide, namely the nucleotide modified with 5'-(E)-vinyl-2'-methoxy-modified phosphonate group (5'-(E)-VP-2'-OMe), is as shown in formula (11).

[0172] In one embodiment of the present invention, the 5' end and / or 3' end of the positive strand are further connected with an invab.

[0173] In some implementations, when invab is attached to the 5' end of the justice chain, the structure is as follows: It can also be modified with thiophosphates (invab): When invab is connected at the 3' end of the justice chain, it is as follows: When L96 is coupled at the 3' end, the following formula is shown:

[0174] In the siRNA preparation method described in this invention, unless otherwise specified, the nucleoside monomer refers to the modified or unmodified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the desired siRNA. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for siRNA synthesis. All nucleoside monomers used in this invention are commercially available.

[0175] This invention provides a novel double-stranded RNA molecule targeting CFB, specifically siRNA. In vitro and in vivo experiments demonstrate that the siRNA provided by this invention can effectively inhibit CFB expression, with an inhibition rate exceeding 70%.

[0176] Based on the mechanism of action of siRNA, the CFB-targeting siRNA provided in this invention, upon entering the cell, mediates the formation of an siRNA silencing induction complex (RISC). The activated RISC, through base pairing, localizes to a specific site on the CFB mRNA, causing efficient degradation of the CFB mRNA and thus significantly reducing CFB protein levels. Therefore, the siRNA provided in this invention can be used for the prevention and treatment of diseases related to CFB overexpression or abnormal activation. Detailed Implementation

[0177] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0178] definition

[0179] The abbreviations for nucleotide monomers in nucleic acid sequences are shown in Table 1. It is important to note that when these monomers are present in oligonucleotides, they are interconnected via 5'-3'-phosphodiester bonds or 5'-3'-thiophosphodiester bonds.

[0180] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0181] Table 1. Abbreviations and explanations of nucleotide monomers in nucleic acid sequences.

[0182] In the context of this disclosure, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the strand can be inferred from the sequence of its complementary strand. When hypoxanthine (I) is artificially introduced, it mainly pairs with the purine bases guanine (C), adenine (A), and pyrimidine (T) (or uracil (U) in RNA).

[0183] In the context of this disclosure, particularly in describing methods for preparing siRNA, pharmaceutical compositions, or siRNA conjugates of the present invention, unless otherwise specified, a nucleoside monomer refers to a modified or unmodified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. The nucleoside monomers used in the embodiments of this disclosure are commercially available.

[0184] In the context of this disclosure, "coupling" refers to the covalent connection between two or more chemical parts, each with a specific function; correspondingly, "coupling" refers to a compound formed by the covalent connection between these chemical parts. Further, "siRNA conjugate" refers to a compound formed by the covalent attachment of one or more chemical parts with specific functions to siRNA. siRNA conjugates should be understood, depending on the context, as a collective term for multiple siRNA conjugates or a siRNA conjugate represented by a specific chemical formula. In the context of this disclosure, "coupling molecule" should be understood as a specific compound that can be reactively coupled to siRNA to ultimately form the siRNA conjugate of this disclosure.

[0185] In the context of this disclosure, the terms “treatment,” “relief,” or “improvement” are used interchangeably herein. These terms refer to methods of achieving beneficial or desired outcomes, including, but not limited to, treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0186] In this disclosure, complementary sequences within a double-stranded RNA molecule comprise base pairings along the entire length of the sense and antisense strands. Such sequences may be referred to as “perfectly complementary” to each other in the application. However, when the sense strand is referred to herein as “fundamentally complementary” or “partially complementary” relative to the antisense strand, the two sequences may be perfectly complementary, or they may form one or more mismatched base pairs, such as 1, 2, 3, 4, or 5 mismatched base pairs, but preferably no more than 5, while maintaining the ability to hybridize under the conditions most relevant to its final application. In determining complementarity, overhangs should not be considered mismatches. For example, a double-stranded RNA molecule comprising a 19-nucleotide sense strand and a 21-nucleotide antisense strand, wherein the longer nucleotide contains a 19-nucleotide sequence perfectly complementary to the shorter nucleotide, may still be referred to as “perfectly complementary.”

[0187] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0188] Example 1: Preparation of siRNA

[0189] 1. CFB mRNA

[0190] CFB mRNA refers to mRNA with the sequence shown in GeneBank registration numbers NM_001710.6, XM_005553440.3, NM_212466.3, or NM_008198.3. The siRNA sequence used in the experiment was synthesized by Suzhou Genecast Co., Ltd., and stored at -20℃.

[0191] 2. siRNA sequence

[0192] siRNAs were designed based on the NM_001710.6 transcript of CFB mRNA, and their sequences are shown in Table 2.

[0193] Table 2. Unmodified siRNA

[0194] In Table 2 above, column 1 is the name of the CFB siRNA, column 2 is the positive strand of the CFB siRNA, column 4 is the negative strand of the CFB siRNA, column 3 is the sequence number of the positive strand of the CFB siRNA in column 2, and column 5 is the sequence number of the negative strand of the CFB siRNA in column 4.

[0195] The CFB siRNA sequences shown in Table 2 were modified using the M1 modification pattern. The M1 modification pattern involves 2'-fluorination of nucleotides 7, 8, and 9 on the sense strand, and 2'-methoxyination of the remaining nucleotides, from the 5' end to the 3' end. Similarly, 2'-fluorination of nucleotides 2, 6, 14, and 16 on the antisense strand, and 2'-methoxyination of the remaining nucleotides, are performed. Phosphothioester linkages are formed between nucleotides 1 and 2, and between nucleotides 2 and 3 on the sense strand, and between nucleotides 1 and 2, 2 and 3, 19 and 20, and 20 and 21 on the antisense strand, resulting in the modified CFB siRNA. The modified siRNA is named according to "name of the original siRNA" + "modification pattern". For example, the modified hC-by502 obtained by modifying the sense and antisense strands of hC-by502 with M1 is named "hC-by502M1" or simply "502M1".

[0196] M1 Modification Mode:

[0197] Chain of Justice 5'-3':

[0198] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0199] Antisense chain 5'-3':

[0200] NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm

[0201] Where N represents a nucleotide (A, U, G, C or I), m indicates that the nucleotide to the left of m is a nucleotide modified with 2'-methoxy, s indicates a thiophosphate group modification, that is, the two nucleotides adjacent to the letter s are connected by a thiophosphate group, and f indicates that the nucleotide to the left of f is a nucleotide modified with 2'-fluoride.

[0202] Table 3. Exemplary M1-modified siRNAs

[0203] Example 2: Detection of siRNA activity in inhibiting CFB gene expression

[0204] (I) Using the psiCHECK vector to detect the knockdown activity of siRNA on CFB mRNA expression

[0205] Use psiCHECK TM -2 vectors were used to construct detection plasmids for the siRNAs provided in this disclosure.

[0206] In psiCHECK TM In the -2 vector, the firefly luciferase gene served as an internal reference reporter gene for normalization; the *Rhizopus luciferase* gene served as the master reporter gene for monitoring the regulatory activity of siRNA. In this experiment, the CFB gene fragment was cloned into psiCHECK. TM The siRNA was used to obtain a detection plasmid by identifying a multiple cloning site downstream of the translation stop codon for Renal luciferase in the vector. When the siRNA binds to the CFB mRNA transcribed from this plasmid, it leads to the degradation of the Renal luciferase-CFB fusion mRNA, thereby decreasing Renal luciferase expression. The strength of the siRNA's knockdown activity on CFB mRNA expression can be determined by detecting the degree of decrease in the amount of luminescent products catalyzed by Renal luciferase.

[0207] 1. Based on psiCHECK TM Construction of plasmid for detecting -2

[0208] Seven different fragments of the hCFB gene were cloned into psiCHECK. TM The Xho I / Not I sites of the -2 vector were used to obtain 7 detection plasmids, as shown in Table 4.

[0209] Table 4. Detection plasmids

[0210] 2. Cell culture and transfection

[0211] Add 5 μL of siRNA to each well of a 96-well plate (the final concentration of siRNA in each well will be 1 nM, 0.1 nM, or 0.01 nM), followed by 12.5 μL of Opti-MEM (containing any one of the seven detection plasmids shown in Table 4, 20 ng), then add a mixture of 32.5 μL of Opti-MEM and 0.3 μL of Lipofectamine 2000 (purchased from Invitrogen, catalog number 11668-019); mix at room temperature and let stand for 15 minutes. Afterward, add 1 × 10⁻⁶ ppm of siRNA to each well. 6 50 μL of DMEM medium suspension (complete DMEM medium, purchased from Transgen Biotech, catalog number FI101-01) was added to each HEK293T cell, and the cell plate was then incubated in a 37°C incubator with 5% CO2 and 95% air for 24 hours. In addition, a Mock group (solvent control group) was set up, in which 5 μL of DEPC water was added to replace the siRNA; and an NC group (siRNA with meaningless sequences) was set up.

[0212] 3. Dual-luciferase assay

[0213] After 24 hours of culture, remove the cell plate, aspirate 20 μL of culture medium from each well, and then add 75 μL of [unspecified ingredient] to each well. Luciferase Reagent was used, and the cell plates were then incubated at 30 rpm for 20 minutes at room temperature to induce lysis. The lysis buffer was then thoroughly mixed, and 75 μL was transferred to each well of the cell assay plate. The Firefly fluorescence values ​​were read using a microplate reader. Then, 75 μL of Luciferase Reagent was added to each well of the assay plate. Stop & Reagent was used to detect the fluorescence value of Renilla using a microplate reader.

[0214] The Renilla fluorescence values ​​in each well were normalized using the Firefly fluorescence values, with the formula R = Renilla fluorescence value / Firefly fluorescence value. The fluorescence ratio for each test group or solvent control group was the average of the fluorescence ratios of its three culture wells. Compared with the control group, the knockdown degree of CFB mRNA expression level by each siRNA was expressed as a percentage (inhibition rate), with inhibition rate % = (1 - R). 测试组 / R MOCK )×100%.

[0215] The inhibition rates of the siRNAs provided in this disclosure are shown in Tables 5 to 11.

[0216] Table 5. Inhibition rate of siRNA (plasmid psiCHECK2) TM -hCFB-1)

[0217] Table 6. Inhibition rate of siRNA (plasmid psiCHECK2) TM -hCFB-2)

[0218] Table 7. Inhibition rate of siRNA (plasmid psiCHECK2) TM -hCFB-3)

[0219] Table 8. Inhibition rate of siRNA (plasmid psiCHECK2) TM -hCFB-4)

[0220] Table 9. Inhibition rate of siRNA (plasmid psiCHECK2) TM -hCFB-5)

[0221] Table 10. Inhibition rate of siRNA (plasmid psiCHECK2) TM -hCFB-6)

[0222] Table 11. Inhibition rate of siRNA (plasmid psiCHECK2) TM -hCFB-7)

[0223] (II) Detection of the knockdown activity of siRNA on CFB mRNA expression levels using CFB-expressing cells (single-concentration experiment)

[0224] 1. Construction of stable CFB lentivirus cells

[0225] HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Stable transfected CFB lentivirus cells, HepG2 CFB LV, were constructed using the following methods:

[0226] HepG2 cells were digested with trypsin, resuspended in complete culture medium, and the cell concentration was adjusted to 6 × 10⁶ cells / year. 4Cells were cultured at a concentration of 100 μL / mL, and then 500 μL was seeded into each well of a 24-well plate. After 12-20 hours of seeding, 100 μL of lentivirus expressing CFB (purchased from Suzhou Gemma Gene Co., Ltd., CFB transcription number NM_001710.6) was added to each well, and the medium was replaced with fresh medium after 12-20 hours. After 72 hours, puromycin was added to each well to a final concentration of 2 μg / mL, and the medium was replaced with fresh medium containing 2 μg / mL puromycin every 2-3 days. After 48 hours of puromycin selection, cell samples were collected, RNA was extracted, and validated by RT-PCR.

[0227] The culture conditions for HepG2 CFB LV were MEM + 10% FBS + 1% P.S + 1% sodium pyruvate + 1% NEAA + 1ug / mL puromycin.

[0228] 2. siRNA transfection of cells

[0229] Cultured HepG2 CFB LV cells were routinely digested with trypsin, resuspended in complete culture medium, and diluted to 6 × 10⁶. 5 The sample was inoculated at 50 μL / well in a 96-well plate for subsequent experiments.

[0230] The siRNA test group was set up with a final transfection concentration of 1 nM, using Thermo Fisher Scientific's Lipofectamine transfection reagent. TM RNAiMAX was used to transfect HepG2 CFB LV cells in the 96-well plates with siRNA according to the manufacturer's instructions. Forty-eight hours after transfection, the cell culture medium in each well was discarded, and 50 μL of cell lysis buffer (Germage) was added to each well. After incubation for 5 minutes for complete lysis, 5 μL of stop solution was added to each well to obtain the final lysis product. In addition, NC, MOCK, and BLANK groups were set up as controls; the MOCK group contained no siRNA in the cell wells; the BLANK group contained only cells; and the NC group contained siRNA with a meaningless sequence.

[0231] 3. RT-qPCR detection

[0232] The cell lysis products obtained from the above groups were added to DNase I to remove genomic DNA, and RNA templates were obtained after treatment. RT-qPCR was performed using GAPDH as an internal reference gene and the primers and probes shown in Table 12.

[0233] Table 12. Primers and probes used for RT-qPCR

[0234] Amplification was performed according to the standard RT-qPCR procedure, and fluorescence was collected. The relative quantification of the target gene CFB in each test group was calculated using the Ct(ΔΔCt) method, as follows:

[0235] ΔCt(test group) = Ct(test group) 目标基因 –Ct(test group) 内参基因 )

[0236] ΔCt(control group) = Ct(control group) 目标基因 )–Ct(control group) 内参基因 )

[0237] ΔCt(test group) = ΔCt(test group) - ΔCt(control group) 平均 )

[0238] ΔCt(control group) = ΔCt(control group) - ΔCt(control group) 平均 )

[0239] Among them, ΔCt (control group) 平均 ) is the arithmetic mean of ΔCt (control group) for each sample in the control group (i.e., the Mock group); thus, each sample in the test group and the control group corresponds to a ΔCt value.

[0240] Using the control group as a baseline, the expression level of CFB mRNA in the test group was normalized, and the expression level of CFB mRNA in the control group was defined as 100%.

[0241] The relative expression level of CFB mRNA in the test group = 2 - ΔΔCt(test group) × 100%

[0242] The inhibition rate (100% - relative expression level) was further calculated, and the results are shown in Table 13.

[0243] Table 13. Inhibition rate of siRNA against CFB mRNA

[0244] (III) Detection of siRNA knockdown activity on CFB mRNA expression levels using CFB-expressing cells (three-concentration experiment)

[0245] Based on the detection results shown in Part (II) of this embodiment, 143 siRNAs were selected. Further referring to the description in that part, the 143 siRNAs were transfected into HepG2 CFB LV cells at final concentrations of 1 nM, 0.1 nM and 0.01 nM, respectively. The inhibition rate (100% - relative expression level) was detected and calculated. The results are shown in Table 14.

[0246] Table 14. Inhibition rate of siRNA against CFB mRNA

[0247] (iv) IC50 of siRNA knockdown of CFB mRNA expression level

[0248] Based on the detection results shown in Part (III) of this embodiment, the siRNA with better performance was selected. Further referring to Part (II), the siRNA was transfected into HepG2 CFB LV cells. The difference was that the final concentration of siRNA at the time of transfection was 10 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, 0.012 nM, 0.004 nM, and 0.001 nM.

[0249] Using the log value of siRNA concentration as the X-axis and the inhibition rate as the Y-axis, the dose-response curve was fitted using the analysis software GraphPadPrism8 to obtain the IC50 value of each siRNA.

[0250] The IC50 values ​​for knocking down CFB mRNA expression levels with different siRNAs in HepG2 CFB LV cells are shown in Table 15.

[0251] Table 15. IC50 of siRNA inhibiting CFB mRNA levels (Test 1)

[0252] Example 3: In vivo mouse experiment on the effect of siRNA in inhibiting CFB gene expression.

[0253] (I) Experiment on the silencing effect of siRNA on CFB mRNA

[0254] A single-concentration administration experiment was conducted in hCFB transgenic mice using siRNAs with good efficacy. These transgenic mice were constructed by Suzhou Gemma Gene Co., Ltd.

[0255] Dilute siRNA with 1 OD / 40 μL of physiological saline. One mouse (female or male) per group. The dosage for mice is 3 mg / kg based on the mass of siRNA / mouse body weight, and the administration volume is 100 μL (physiological saline or physiological saline dilution of siRNA). Subcutaneous injection is performed on day 0.

[0256] On day 14 after administration, the mice were euthanized, and a piece of liver tissue was taken, 20 mg per piece, cut into small pieces, and placed in RNA protection solution overnight at -4°C. The next day, it was placed at -20°C.

[0257] For RT-qPCR detection, DNase I was added to the liver tissue lysate to remove genomic DNA, and the resulting RNA template was obtained. Following the description in Part (II) of Example 2, GAPDH was used as an internal reference gene to detect the in vivo inhibition rate of each siRNA on CFB mRNA expression levels. The results are shown in Table 16.

[0258] Table 16. In vivo inhibition rate of siRNA against CFB mRNA

[0259] (II) In vivo gene expression knockdown effect experiment

[0260] As described in Part (I) of this embodiment, 20 siRNAs were selected, and the activity of siRNAs in inhibiting CFB gene expression was tested again in hCFB transgenic mice.

[0261] Similarly, the siRNA to be tested was diluted with 1 OD / 40 μL of physiological saline. Mice were grouped as follows: 5 mice in the physiological saline group and 3 mice in each siRNA test group. The dosage for mice was 3 mg / kg, and the administration volume was 100 μL (physiological saline or physiological saline dilution of siRNA), administered subcutaneously on the same day.

[0262] On day 14 after administration, the mice were euthanized, and a piece of liver tissue was taken, 20 mg per piece, cut into small pieces, and placed in RNA protection solution overnight at -4°C. The next day, it was placed at -20°C.

[0263] For RT-qPCR detection, DNase I was added to the liver tissue lysate to remove genomic DNA, and the resulting RNA template was obtained. Following the description in Part (II) of Example 2, GAPDH was used as an internal reference gene to detect the in vivo inhibition rate of each siRNA on CFB mRNA expression levels. The results are shown in Table 17.

[0264] Table 17. In vivo inhibition rate of siRNA against CFB mRNA

[0265] Example 4: Synthesis of siRNA with different modification modes

[0266] Furthermore, the high-performing CFB siRNAs were modified using other modification modes, such as M2. The modified siRNAs were named according to "original siRNA name" + "modification mode." For example, the modified hC-by502, obtained by modifying the sense and antisense strands of hC-by502 with M2, was named "hC-by502M2" or simply "502M2." The M2, M4, and M6 modification modes are as follows.

[0267] M2:

[0268] Chain of Justice 5'-3':

[0269] NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0270] Antisense chain 5'-3':

[0271] NmsNfsNmNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmsNmsNm

[0272] M4:

[0273] Chain of Justice 5'-3':

[0274] NmsNmsNfNmNfNmNfNfNfNmNfNmNfNmNfNmNfNmNf

[0275] Antisense chain 5'-3':

[0276] NmsNfsNmNfNmNfNmNfNmNfNmNmNmNfNmNfNmNfNmsNmsNm

[0277] M6:

[0278] Chain of Justice 5'-3':

[0279] (N-LNA)NmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm

[0280] Antisense chain 5'-3':

[0281] NmsNfsNmNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmsNmsNm

[0282] Where N represents a nucleotide (A, U, G, C, or I), m indicates that the nucleotide to the left of m is a 2'-methoxy modified nucleotide, s indicates a phosphate thioester group modification, meaning that the two nucleotides adjacent to the letter s are linked by a phosphate thioester group, f indicates that the nucleotide to the left of f is a 2'-fluoro modified nucleotide, and (LNA) indicates that the nucleotide to the left is a locked nucleic acid modified as follows:

[0283] Exemplary siRNAs prepared are shown in Table 18 below.

[0284] Table 18. Exemplary modified siRNAs

[0285] Based on other modification patterns such as M1, M2, M4, M6, and the modified siRNAs shown in the table above, further modification with 5'-(E)-VP is performed at the 5' end of the antisense strand to obtain 5'-(E)-VP modified siRNA.

[0286] For example, based on the M1 modification pattern, the modification pattern of siRNA further modified at the 5' end of the antisense strand with 5'-(E)-VP is as follows:

[0287] M1VP:

[0288] Chain of Justice 5'-3':

[0289] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm

[0290] Antisense chain 5'-3':

[0291] VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm

[0292] The siRNAs modified by M1VP are named according to the "name of the siRNA before modification" + "modification pattern". For example, the modified hC-by502 obtained by modifying the sense and antisense strands of hC-by502 with M1VP is named hC-by502M1VP.

[0293] Exemplary siRNAs with 5'-(E)-VP modification at the 5' end of the antisense strand are shown in Table 19 below.

[0294] Table 19. Exemplary modified siRNAs

[0295] Example 5: L96 conjugation of siRNA

[0296] Furthermore, L96 was coupled to the 3' end of the positive strand of the modified siRNA.

[0297] For example, based on the M1 modification pattern, the modification pattern of L96-coupled siRNA is as follows:

[0298] M1G:

[0299] Chain of Justice 5'-3':

[0300] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96

[0301] Antisense chain 5'-3':

[0302] NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm

[0303] For example, based on the M1VP modification mode, the modification mode of L96-coupled siRNA is as follows:

[0304] M1GVP:

[0305] Chain of Justice 5'-3':

[0306] NmsNmsNmNmNmNmNfNfNfNmNmNmNmNmNmNmNmNmNm-L96

[0307] Antisense chain 5'-3':

[0308] VPNmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm

[0309] N represents a nucleotide (A, U, G, C, or I), m indicates that the nucleotide to the left of m is a nucleotide modified with 2'-methoxy, s indicates a thiophosphate group modification, that is, the two nucleotides adjacent to the letter s are connected by a thiophosphate group, f indicates that the nucleotide to the left of f is a nucleotide modified with 2'-fluoride, and VP indicates that the 5' end of the nucleotide is modified with (E)-vinyl phosphate.

[0310] The siRNAs modified by M1GVP are named according to the "name of the siRNA before modification" + "modification pattern". For example, the modified hC-by502 obtained by modifying the sense and antisense strands of hC-by502 with M1GVP is named hC-by502M1GVP.

[0311] Using different modification modes, siRNAs further modified with VP and siRNAs conjugated with L96 were prepared. Some of the prepared siRNAs are shown in Table 20.

[0312] Table 20. Exemplary VP-modified siRNAs and L96-coupled siRNAs

[0313] Example 6. Study on the activity of siRNA silencing the CFB gene with different modification modes

[0314] (I) IC50 of siRNA knockdown of CFB mRNA expression levels with different modification modifiers

[0315] Referring to the method described in Part (II) of Example 2, siRNA was transfected into HepG2 CFB LV, except that the final concentration of siRNA at the time of transfection was 10 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, 0.012 nM, 0.004 nM, and 0.001 nM.

[0316] The IC50 values ​​of each siRNA were obtained by fitting dose-response curves using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis. The results are shown in Table 21.

[0317] Table 21. IC50 of siRNA knockdown of CFB mRNA expression level

[0318] (II) Experiment on the silencing effect of siRNA on CFB mRNA

[0319] Similarly, the siRNA to be tested was diluted with 1 OD / 40 μL of physiological saline. Mice were grouped as follows: at least 3 mice in each saline group and each siRNA test group. The dosage for mice was 1 mg / kg, and the administration volume was 100 μL (physiological saline or physiological saline dilution of siRNA), administered subcutaneously on the same day.

[0320] On day 14 after administration, a total of 51 mice were euthanized, and a piece of liver tissue was taken, 20 mg per piece, cut into small pieces, and placed in RNA protection solution overnight at -4°C. The next day, it was placed at -20°C.

[0321] For RT-qPCR detection, DNase I was added to the liver tissue lysate to remove genomic DNA, and RNA templates were obtained after treatment. Following the description in Part (II) of Example 2, GAPDH was used as an internal reference gene to detect the relative expression level and inhibition rate of CFB mRNA after administration of each siRNA. The results are shown in Table 22.

[0322] Table 22. In vivo inhibition rate of siRNA against CFB mRNA

[0323] Example 7: In vivo mouse experiment on the effect of siRNA in inhibiting CFB gene expression.

[0324] As described in Part (a) of Example 3, the activity of L96- and VP-modified siRNA in inhibiting CFB gene expression was detected in hCFB transgenic mice.

[0325] Similarly, the siRNA to be tested was diluted with 1 OD / 40 μL of physiological saline. Mice were grouped as follows: at least 9 mice in each saline group and each siRNA test group. The dosage for mice was 1 mg / kg, and the administration volume was 200 μL (physiological saline or physiological saline dilution of siRNA), administered subcutaneously on the same day. A model group (NA group) was also set up.

[0326] Mice were euthanized 6 days before drug administration (NA group), on the day of drug administration (NA group), and on days 14, 21, and 28 after drug administration. After euthanasia, a 20mg piece of liver tissue was taken, shredded, and placed in RNA protection solution overnight at -4°C, and then placed at -20°C the next day.

[0327] For RT-qPCR detection, DNase I was added to the liver tissue lysate to remove genomic DNA, and RNA templates were obtained after treatment. Following the description in Part (II) of Example 2, GAPDH was used as an internal reference gene to detect the relative expression levels of CFB mRNA after administration of each siRNA. The results are shown in Table 23.

[0328] Table 23. Relative expression levels of CFB mRNA after siRNA administration

[0329] Example 8. In vivo pharmacological activity of siRNA in CFB homozygous mice

[0330] The siRNA with better efficacy was selected for experiments in CFB homozygous mice. Three C57BL / 6J-CFB- / - mice (human CFB transgenic mice, male or female, 6-8 weeks old) were used in each group. The mice were administered siRNA subcutaneously once at a dose of 1 mg / kg (siRNA mass / mouse body weight) or 200 μL of saline per mouse. Before administration (day 0), 80 μL of serum was obtained by centrifugation from the orbital vein of each mouse; serum was obtained again on day 14 after administration. Serum samples were used for Western blotting (WB) to examine changes in serum hCFB protein levels on day 14 after siRNA administration compared to day 0 before administration.

[0331] In this Western blotting (WB) assay, mALB was used as an internal control protein; WB detection was performed using conventional methods in the art. ImageJ was used to calculate the grayscale values ​​of the bands in the developed image, and the ratio of hCFB / mALB grayscale values ​​was used as the hCFB protein content in the sample. Then, the relative expression level (i.e., residual activity) of hCFB was calculated using the following formula:

[0332] The relative expression level of hCFB was calculated as the ratio of hCFB protein content on day 14 to hCFB protein content on day 0. Results are shown in Table 24.

[0333] Table 24. Knockdown activity of siRNA on CFB protein expression in CFB homozygous mice

[0334] *: AD-1725763 originates from patent US11,965,166B2, with the following sequence:

[0335] Chain of Justice (5' to 3'): GmsAmsAmUmUmCmCmUmGfAfAfUmUmUmUmAmUmGmAmCmUm-L96;

[0336] Antisense chain (5' to 3'):

[0337] Ams(dG)sUmCm(dA)Um(dA)AmAmAmUm(dT)CmAfGmGmAmAmUmUmCmsCmsUm

[0338] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An siRNA comprising a sense strand and an antisense strand: the sense strand comprises a nucleotide sequence as shown in any odd-numbered n of SEQ ID NO. 1 to SEQ ID NO. 394, and the antisense strand comprises a nucleotide sequence as shown in any even-numbered n+1 of SEQ ID NO. 1 to SEQ ID NO. 394, wherein n is an odd number between 1 and 394.

2. The siRNA of claim 1, wherein At least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.

3. The siRNA of claim 1, wherein the modified nucleotide is selected from at least one of the following group: a 2'-methoxy modified nucleotide, a 2'-fluoro modified nucleotide, a deoxy-nucleotide, a 2'-deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a restricted ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a nucleotide comprising a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a glycerol nucleotide (GNA), an unlocked nucleotide acid (UNA), a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

4. The siRNA according to any one of claims 1 to 3, characterized in that, The siRNA is further 5'-(E)-VP modified at the 5' end of the antisense strand.

5. The siRNA according to any one of claims 1 to 4, characterized in that, The siRNA is further linked with an inverted abasic nucleotide (invab) at the 5' end and / or 3' end of the sense strand.

6. The siRNA according to any one of claims 1 to 5, characterized in that, The siRNA comprises a sense strand and an antisense strand as shown in: (1) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO. 409, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO. 430; Sense strand: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense strand: VPAmsGfsUmCmAmUmAmAmAfAmUmUfCmAfGmGfAmAmUmsUmsCm (SEQ ID NO. 430); (2) the sense strand comprises a nucleotide sequence as shown in SEQ ID NO. 409, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO. 412; Sense strand: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); (3) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 401; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); (4) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 402; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); (5) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 403; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmAfUmC(d)AmCfUmCf(Im)CmAmsUmsUm (SEQ ID NO. 403); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); (6) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 404; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Antisense: AmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 401); (7) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 405; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); antisense: AmsGfsAmGmAmUmCmUfCmAfUmC(d)AmCfUmCf(Im)CmAmsUmsUm (SEQ ID NO. 405); (8) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 406; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); antisense: AmsGfsAmGfAmUmCmUmCmAmUmCfAmCfUmCf(Im)CmAmsUmsUm (SEQ ID NO. 406); (9) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 407, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 408; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGmAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 407); antisense: AmsGfsAmGmAmUmCmUmCmAmUmC(d)AmCfUmCf(Im)CmAmsUmsUm (SEQ ID NO. 408); (10) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 397, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 398; Sense: AmsUmsUmCmCmUmGfAfAfUmUmUmUmAmUmGmAmCmUm (SEQ ID NO. 397); antisense: AmsGfsUmCmAmUfAmAmAmAmUmUmCmAfGmGfAmAmUmsUmsCm (SEQ ID NO. 398); (11) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 410; Sense: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); antisense: AmsGfsUmCmAmUmAmAfAm(Im)UmUfCmAfGmGfAmAmUmsUmsCm (SEQ ID NO. 410); (12) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 411; sense: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); antisense: AmsGfsUmCmAmUmAmAmAfAmUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 411); (13) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 395, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 396; sense: UmsGmsUmGmAmGmUfGfAfUmGmAmGmAmUmCmUmCmUm (SEQ ID NO. 395); antisense: AmsGfsAmGmAmUfCmUmCmAmUmCmAmCfUmCfAmCmAmsUmsUm (SEQ ID NO. 396); (14) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 413; sense: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); antisense: AmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 413); (15) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 414; sense: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); antisense: AmsGfsUmCmAmUfAmAmAmA(d)UmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 414); (16) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 415; sense: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); (19) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 419; sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); antisense: VPAmsGfsAmGmAmUfCmUmCmAmUmCmAmCfUmCfAmCmAmsUmsUm (SEQ ID NO. 419); (20) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 420; sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); antisense: VPAmsGfsAmGmAmUfCmUmCmA(d)UmCfAmCf(Im)CfAmCmAmsUmsUm (SEQ ID NO. 420); (21) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 399, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO. 421; sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); antisense: VPAmsGfsAmGmAmUfCmUmCmAmUmCmAmCfUmCfAmCmAmsUmsUm (SEQ ID NO. 421); ​ ​ ​ ​ Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); (22) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 399, and the anti sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 422; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); (23) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 399, and the anti sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 423; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); (24) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 399, and the anti sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 424; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); (25) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 399, and the anti sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 425; Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); (26) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 407, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 426; sense: (Invab) sAmAmUmGmUmGmAfGmUfGmAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 407); antisense: AmsGfsAmGmAmUmCmUmCmAmUmC(d)AmCfUmCf(Im)CmAmsUmsUm (SEQ ID NO. 426); (27) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 397, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 427; sense: AmsUmsUmCmCmUmGfAfAfUmUmUmUmAmUmGmAmCmUm (SEQ ID NO. 397); antisense: VPAmsGfsUmCmAmUfAmAmAmAmUmUmCmAfGmGfAmAmUmsUmsCm (SEQ ID NO. 427); (28) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 428; sense: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); antisense: VPAmsGfsUmCmAmUmAmAfAm(Im)UmUfCmAfGmGfAmAmUmsUmsCm (SEQ ID NO. 428); (29) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 429; sense: (Invab) sGmAmAmUmUmCmCfUmGfAfAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); antisense: VPAmsGfsUmCmAmUmAmAmAfAmUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 429); (30) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO. 399, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO. 400; sense: (Invab) sAmAmUmGmUmGmAfGmUfGfAfUmGmAmGmAmUmCmUmCmUms (Invab) (SEQ ID NO. 399); Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); (31) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 431; Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); (32) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 432; Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); (33) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 409, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 433; Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); (34) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO. 416, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO. 434; Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); Sense: (Invab) sGmAmAmUmUmCmCfUmGfAmAfUmUmUmUmAmUmGmAmCmUms (Invab) (SEQ ID NO. 409); Antisense: VPAmsGfsUmCmAmUmAmAmAmAfUmUfCmAf(Im)GfAmAmUmsUmsCm (SEQ ID NO. 431); 7. The siRNA according to any one of claims 1 to 6, wherein the 3 '-end or 5 '-end of the sense strand of the siRNA or any nucleotide in the sense strand is further linked with a ligand.

8. The siRNA of claim 7, wherein the ligand comprises N-acetylgalactosamine (GalNAc) or a derivative thereof. the ligand comprises N-acetylgalactosamine (GalNAc) or a derivative thereof.

9. The siRNA according to any one of claims 7 to 8, characterized in that, The ligand is L96 of the formula:

10. A composition comprising the siRNA of any one of claims 1 to 9.

11. A cell comprising the siRNA of any one of claims 1 to 9.

12. Use of the siRNA of any one of claims 1 to 9, the composition of claim 10, or the cell of claim 11 in the manufacture of a medicament for treating a disease caused by abnormal expression of CFB.

13. A method of treating a disease caused by abnormal expression of CFB, the method comprising administering to a subject in need thereof the siRNA of any one of claims 1 to 9, the composition of claim 10, or the cell of claim 11.

14. A method of inhibiting CFB expression in a cell, the method comprising: (a) contacting the cell with the siRNA of any one of claims 1 to 9, the composition of claim 10, or the cell of claim 11, (b) maintaining the cell resulting from step (a) for a time sufficient to achieve degradation of mRNA transcripts of the CFB gene to effect inhibition of CFB expression in the cell.

15. The use of claim 12 or the method of any one of claims 13 to 14, wherein the disease caused by abnormal expression of CFB is a disease caused by upregulation of CFB expression; or comprises a disease associated with abnormal activation of the complement alternative pathway. or the disease caused by the abnormal expression of CFB includes, but is not limited to, nervous system diseases Alzheimer's disease (AD), autoimmune neurological diseases, neuromyelitis optica (NMO), myasthenia gravis (gMG), etc., eye diseases such as age-related macular degeneration, age-related macular degeneration (AMD), geographic atrophy, uveitis, glaucoma, kidney diseases (such as atypical hemolytic uremic syndrome, C3 glomerulopathy, IgA nephropathy, atypical hemolytic uremic syndrome, membrane proliferative glomerulonephritis, lupus nephritis), hematological diseases, cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, thrombotic microangiopathy, sepsis / septic shock, diabetic retinopathy, retinitis pigmentosa, macular edema, Behcet uveitis, multifocal choroiditis, Fukaya-Ogata syndrome, intermediate uveitis, birdshot retinochoroiditis, sympathetic ophthalmia, ocular cicatricial pemphigoid, ocular pemphigus, non-arteritic ischemic optic neuropathy, postoperative inflammation, retinal vein occlusion, neurological disorders, multiple sclerosis, stroke, Guillain-Barre syndrome, traumatic brain injury, Parkinson's disease, inappropriate or undesirable complement activation disorders, hemodialysis complications, hyperacute allograft rejection, xenograft rejection, interleukin-2-induced toxicity during IL-2 therapy, inflammation of autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, myocarditis, post-ischemic reperfusion disorders, myocardial infarction, balloon angioplasty, post-pump syndrome in cardiopulmonary bypass or renal bypass surgery, atherosclerosis, hemodialysis, renal ischemia, aortic reconstruction, mesenteric arterial reperfusion after infectious disease or sepsis, immune complex disorders and autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, liver fibrosis, hemolytic anemia, myasthenia gravis, tissue regeneration, nerve regeneration, dyspnea, hemoptysis, ARDS, asthma, chronic obstructive pulmonary disease (COPD), pulmonary emphysema, pulmonary embolism and infarction, fibrosis-causing dust diseases, pulmonary fibrosis, asthma, allergy, bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, Goodpasture's syndrome, pulmonary vasculitis, oligoimmune vasculitis, immune complex-related inflammation, antiphospholipid syndrome, glomerulonephritis and obesity, etc.

16. Use of the siRNA of any one of claims 1 to 9, the composition of claim 10, or the cell of claim 11 in the manufacture of a medicament for inhibiting the expression of CFB in a cell.

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