Sirna for inhibiting expression of complement component c5, use thereof, and product thereof

Abstract

Provided in the present invention are a siRNA for inhibiting the expression of complement component C5, the use thereof, and a product thereof, belonging to the technical field of small-molecule drugs and relating to the design and use of siRNA for C5 genes. The siRNA of the present invention comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence as shown in SEQ ID NO. 1-SEQ ID NO. 546, and the antisense strand comprises a nucleotide sequence as shown in SEQ ID NO. 547-SEQ ID NO. 1092. Provided in the present invention is a brand new siRNA which can effectively inhibit the expression of complement component C5.

Description

siRNA for inhibiting expression of complement component C5 and application and product thereof

[0001] The present application claims priority to the Chinese patent application No. 2024107330947, filed on June 6, 2024, and entitled "siRNA for inhibiting expression of complement component C5 and application and product thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of small molecule drugs, and relates to the design and application of siRNA, in particular to siRNA for inhibiting expression of complement component C5 and application and product thereof. BACKGROUND

[0003] There are three main pathways of complement activation: (1) classical pathway (CP): refers to the combination of antigen-antibody complexes and C1q, and then the activation of C1r, C1s, C4, and other complement proteins, starting the cascade enzyme reaction process. (2) Lectin pathway: also known as MBL pathway, refers to the recognition and combination of mannose-binding lectin (MBL) in plasma and sugar structure on the surface of pathogenic microorganisms, and the activation of MBL-related proteins, C2, C3, and other factors, starting the cascade enzyme reaction process. (3) Alternative pathway: also known as the bypass pathway, this pathway is activated by complement C3 of pathogenic microorganisms or other exogenous foreign bodies, and then starts the cascade enzyme reaction process under the joint participation of D factor, B factor, and properdin (P factor).

[0004] C5 convertase produced by the above three pathways can cleave C5, initiate common terminal effects, and then exert various biological effects such as phagocytosis, cell lysis, mediation of inflammatory response, regulation of immune response, and clearance of immune complexes.

[0005] In addition to playing a positive protective role, research has found that once the complement system is imbalanced or excessively activated, it can lead to various diseases, such as acute inflammation of eye diseases and periodontal diseases, autoimmune system diseases, tumors, kidney diseases, chronic hemolytic diseases, and neurodegenerative diseases.

[0006] RNAi therapy is a new generation of therapy that acts on mRNA encoding pathogenic proteins. After siRNA enters the cell, it binds to the target mRNA, thereby degrading the mRNA and avoiding the production of functional proteins. Therefore, developing siRNA to inhibit the expression of complement C5 will be an effective way to treat diseases caused by excessive activation of complement. SUMMARY

[0007] The present application provides siRNA for inhibiting expression of complement component C5 and application thereof in preparation of C5 complement targeting inhibitor.

[0008] To achieve the above object, the present application adopts the following technical solutions:

[0009] In one aspect, the present application provides siRNA for inhibiting expression of complement component C5, which comprises a sense strand and an antisense strand forming at least a double-stranded region, wherein the sense strand comprises at least 15 continuous nucleotides differing by no more than 3 nucleotides from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 1-SEQ ID NO. 546; and the antisense strand comprises at least 15 continuous nucleotides differing by no more than 3 nucleotides from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 547-SEQ ID NO. 1092.

[0010] Specifically, at least one nucleotide on the sense strand and / or at least one nucleotide on the antisense strand is a modified nucleotide.

[0011] Specifically, the modified nucleotide comprises a deoxyribonucleotide, a methoxy-modified nucleotide, a fluorine-modified nucleotide or a locked nucleic acid-modified nucleotide.

[0012] Further specifically, the methoxy-modified nucleotide is located in the nucleotide sequence of the antisense strand and the sense strand, and at least the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th nucleotide of the sense strand is a methoxy-modified nucleotide from the 5' end to the 3' end, and at least the 1st, 3rd, 4th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st nucleotide of the antisense strand is a methoxy-modified nucleotide.

[0013] Alternatively, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th nucleotide of the sense strand is a methoxy-modified nucleotide, and at least the 1st, 3rd, 4th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st nucleotide of the antisense strand is a methoxy-modified nucleotide.

[0014] Alternatively, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th position of the nucleotide of the sense strand is a methoxy-modified nucleotide, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st position of the nucleotide of the antisense strand is a methoxy-modified nucleotide.

[0015] Further particularly, the siRNA for inhibiting the expression of complement component C5 according to claim 3 is characterized in that the fluoro-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide in the direction from the 5' end to the 3' end.

[0016] Alternatively, at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 6th, 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide.

[0017] Alternatively, at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 2nd, 6th, 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide.

[0018] Alternatively, at least the 5th, 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 2nd, 6th, 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide.

[0019] Further particularly, the siRNA for inhibiting the expression of complement component C5 according to claim 3 is characterized in that the fluoro-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide in the direction from the 5' end to the 3' end.

[0020] Further particularly, the siRNA for inhibiting the expression of complement component C5 according to claim 3 is characterized in that the fluoro-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide in the direction from the 5' end to the 3' end.

[0021] In yet another aspect, the present application provides an siRNA for inhibiting the expression of complement component C5, characterized in that, in the direction from 5' end to 3' end, the nucleotides at position 1 and 2, and position 2 and 3 of the sense strand are connected by phosphorothioate linkage, the nucleotides at position 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 are methoxy modified nucleotides, and the nucleotides at position 7, 8, 9 are fluorine modified nucleotides; the nucleotides at position 1 and 2, position 2 and 3, position 19 and 20, position 20 and 21 of the antisense strand are connected by phosphorothioate linkage, the nucleotides at position 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 are methoxy modified nucleotides, and the nucleotides at position 2, 6, 14, 16 are fluorine modified nucleotides;

[0022] or the nucleotides at position 1 and 2, and position 2 and 3 of the sense strand are connected by phosphorothioate linkage, the nucleotides at position 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 are methoxy modified nucleotides, and the nucleotides at position 5, 7, 8, 9 are fluorine modified nucleotides; the nucleotides at position 1 and 2, position 2 and 3, position 19 and 20, position 20 and 21 of the antisense strand are connected by phosphorothioate linkage, the nucleotides at position 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 are methoxy modified nucleotides, and the nucleotides at position 2, 6, 14, 16 are fluorine modified nucleotides;

[0023] or the nucleotides at position 1 and 2, and position 2 and 3 of the sense strand are connected by phosphorothioate linkage, the nucleotides at position 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 are methoxy modified nucleotides, and the nucleotides at position 7, 8, 9 are fluorine modified nucleotides; the nucleotides at position 1 and 2, position 2 and 3, position 19 and 20, position 20 and 21 of the antisense strand are connected by phosphorothioate linkage, the nucleotides at position 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 are methoxy modified nucleotides, and the nucleotide at position 2 is deoxynucleotide, and the nucleotides at position 6, 14, 16 are fluorine modified nucleotides;

[0024] or the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions of the sense strand are connected by phosphorothioate group, the nucleotides at the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are methoxy modified nucleotides, the nucleotides at the 7th, 8th, 9th positions are fluoro modified nucleotides; the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, the 20th and 21st positions of the antisense strand are connected by phosphorothioate group, the nucleotides at the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions are methoxy modified nucleotides, the nucleotide at the 6th position is deoxynucleotide, the nucleotides at the 2nd, 14th, 16th positions are fluoro modified nucleotides;

[0025] or the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions of the sense strand are connected by phosphorothioate group, the nucleotides at the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are methoxy modified nucleotides, the nucleotides at the 5th, 7th, 8th, 9th positions are fluoro modified nucleotides; the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, the 20th and 21st positions of the antisense strand are connected by phosphorothioate group, the nucleotides at the 1st, 3rd, 4th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions are methoxy modified nucleotides, the nucleotide at the 5th position is deoxyribonucleotide, the nucleotides at the 2nd, 6th, 14th, 16th positions are fluoro modified nucleotides.

[0026] In another aspect, the present application provides siRNA for inhibiting the expression of complement component C5, wherein the 5' end of the antisense strand comprises at least one modification.

[0027] Specifically, the modification is that the 5' end of the antisense strand comprises at least one 5'-(E)-vinyl phosphonate group modification.

[0028] The present application provides siRNA for inhibiting the expression of complement component C5, wherein the 3' end of the sense strand comprises at least one coupling ligand.

[0029] Specifically, the ligand is GalNAc.

[0030] In another aspect, the present application provides the use of the above-mentioned siRNA in the preparation of a C5 complement targeting inhibitor.

[0031] The C5 complement targeting inhibitor is used for treating diseases caused by complement abnormalities or for improving complement abnormalities caused by diseases.

[0032] Specifically, the C5 complement targeting inhibitor can be used for treating paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome.

[0033] According to the above, the application also provides the use of the siRNA in the preparation of a medicament.

[0034] In another aspect, the application provides a medicament having the function of inhibiting the expression of the complement component C5 gene, characterized in that it comprises any of the above siRNAs.

[0035] The medicament can further comprise other pharmaceutically acceptable adjuvants, including but not limited to excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants.

[0036] Specifically, the excipient is selected from at least one of microcrystalline cellulose, lactose, pregelatinized starch, cyclodextrin, carboxymethyl cellulose, mannitol.

[0037] Specifically, the buffer is selected from at least one of sodium dihydrogen phosphate, sodium bicarbonate, ammonium bicarbonate, sodium acetate, citrate, histidine, succinate.

[0038] Specifically, the emulsifier is selected from at least one of magnesium stearate, zinc stearate, calcium stearate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, glyceryl oleate, polyglyceryl-3 polyricinoleate.

[0039] Specifically, the stabilizer is selected from at least one of acacia gum, agar, alginic acid, cellulose ether, carboxymethyl chitin.

[0040] Specifically, the diluent is selected from at least one of erythritol, mannitol, sorbitol, xylitol, lactose, sucrose, corn starch, potato starch, calcium phosphate, calcium citrate, crystalline cellulose.

[0041] Specifically, the binder is selected from at least one of ethanol, starch paste, sugar syrup, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinyl pyrrolidone.

[0042] Specifically, the preservative is selected from at least one of methylparaben, propylparaben, nipagin, nipasol, nipasol, chlorobutanol, thiomersal, merthiolate, phenoxyethanol, chlorhexidine, benzoic acid, sodium benzoate, chlorocresol, benzalkonium bromide, benzalkonium chloride, hydroxyphenyl ethyl ester.

[0043] Specifically, the lubricant is selected from at least one of magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer.

[0044] In another aspect, the present application provides the use of the aforementioned siRNA in C5 complement detection.

[0045] Specifically, the aforementioned siRNA achieves the binding ability with C5 complement.

[0046] Therefore, the present application simultaneously provides a C5 complement detection kit comprising the aforementioned siRNA, and a C5 complement detection method by using the aforementioned siRNA to bind with the test object and then detecting.

[0047] The C5 complement detection kit can also comprise other common reagents in the art, which can be screened by routine means according to the prior art by those skilled in the art.

[0048] The C5 complement detection method can also comprise other steps, such as sample collection, sample pretreatment, result interpretation, result data analysis, etc., which can be confirmed according to the prior art by those skilled in the art.

[0049] The present application focuses on the detection of C5 complement for non-disease diagnosis function, including but not limited to detection in quality control or quality evaluation.

[0050] In another aspect, the present application provides a treatment method, characterized in that any of the aforementioned siRNA is used.

[0051] In another aspect, the present application provides a genetic engineering product.

[0052] The genetic engineering product comprises, expresses or secretes the aforementioned siRNA.

[0053] The genetic engineering product is selected from an expression vector or a genetically engineered cell.

[0054] Those skilled in the art can select a suitable vector skeleton or cell type according to the actual production needs to achieve the preparation of the aforementioned siRNA.

[0055] Therefore, the present application simultaneously provides a preparation method of the aforementioned siRNA, which can be chemical synthesis or biological synthesis. The biological synthesis generally refers to genetic engineering means, such as transfection of cells by constructing an expression vector for expression, but the scope of protection of the present application is not limited to this form.

[0056] For example, the present application protects a cell comprising any siRNA of the present application and a pharmaceutical composition comprising any siRNA of the present application.

[0057] Compared with the prior art, the present application has the following beneficial effects:

[0058] The application provides non-modified siRNA sequences and modified siRNA sequences targeting C5 complement genes, and experiments prove that the modified siRNA of the application has obvious inhibitory effect on the expression of the complement component C5 gene, and has great clinical significance and popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 is a structure formula of siRNA conjugate formed by GalNAc and siRNA molecules.

[0060] Figure 2 shows the results of the first animal experiment.

[0061] Figure 3 shows the results of the second animal experiment.

[0062] Figure 4 shows the results of the third animal experiment.

[0063] Figure 5 shows the results of the fourth animal experiment.

[0064] Figure 6 shows the results of the fifth animal experiment.

[0065] Figure 7 shows the results of the fifth animal experiment. DETAILED DESCRIPTION

[0066] In the application, the "modified nucleotide" refers to a nucleotide or a nucleotide analog in which the ribosyl 2' position hydroxyl group is replaced by another group, or a nucleotide in which the base is a modified base. The "methoxy modified nucleotide" refers to a nucleotide in which the ribosyl 2'-hydroxyl group is replaced by a methoxy group. The "fluorine modified nucleotide" refers to a nucleotide in which the ribosyl 2' position hydroxyl group is replaced by fluorine. The "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, but is different in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. Such as an isonucleotide, a bridged nucleotide (BNA) or an acyclic nucleotide.

[0067] In one embodiment of the application, the "fluorine modified nucleotide" refers to a nucleotide in which the ribosyl 2' position hydroxyl group is replaced by fluorine, which has the structure shown in the following formula (1). The non-fluorine modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the ribosyl 2' position hydroxyl group is replaced by a non-fluorine group.

[0068] In one embodiment of the present application, the nucleotide formed by substituting the hydroxyl group at the 2' position of the ribose group with a non-fluorine group is well known to those skilled in the art, and the nucleotide can be selected from one of 2'-alkoxy modified nucleotide, 2'-substituted alkoxy modified nucleotide, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-amino modified nucleotide, 2'-substituted amino modified nucleotide, and 2'-deoxy nucleotide.

[0069] In one embodiment of the present application, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, i.e., a methoxy modification, as shown in formula (2); a 2'-substituted alkoxy modified nucleotide, such as a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3); a 2'-amino (2'-NH2) modified nucleotide, as shown in formula (4); and a 2'-deoxy nucleotide (DNA), as shown in formula (5):

[0070] In one embodiment of the present application, the nucleotide analog refers to a group capable of replacing a nucleotide in a nucleic acid, but the structure is different from that of an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide.

[0071] In one embodiment of the present application, the nucleotide analog can be an iso-nucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0072] In one embodiment of the present application, the bridged nucleic acid (BNA) refers to a constrained or inaccessible nucleotide, and the BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar contraction bridged structure, and the bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide.

[0073] In one embodiment of the present application, the BNA can be LNA, ENA, cET BNA, etc., wherein the LNA is as shown in formula (6), the ENA is as shown in formula (7), and the cET BNA is as shown in formula (8):

[0074] In one embodiment of the present application, at least a portion of the phosphate group in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA is a phosphate group with a modification group.

[0075] In one embodiment of the present application, the phosphate group with a modification group is a phosphorothioate group in which at least one of the oxygen atoms in the phosphodiester bond of the phosphate group is replaced by a sulfur atom.

[0076] In one embodiment of the present application, the phosphate group with a modification group is a phosphorothioate group having a structure as shown in formula (9). In one embodiment of the present application, the nucleotide to which the phosphorothioate group is attached has a structure as shown in formula (10), and the phosphorothioate group is attached at at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof.

[0077] In one embodiment of the present application, the VP-modified nucleotide is a vinylphosphonate modification. In one embodiment of the present application, the nucleotide modified with VP and methoxy modification, i.e., 5'-(E)-vinyl-2'-methoxy modified phosphonate group (5'-(E)-VP-2'-OMe) modified nucleotide, has a structure as shown in formula (11). In one embodiment of the present application, the nucleotide modified with VP, methoxy modification and phosphorothioate group modification, i.e., 5'-PS modified nucleotide, has a structure as shown in formula (12).

[0078] In the preparation of siRNA according to the present application, unless otherwise specified, the nucleoside monomer refers to modified or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes also referred to as Nucleoside phosphoramidites) used in the phosphoramidite solid-phase synthesis according to the type and sequence of nucleotides in the siRNA to be prepared. The phosphoramidite solid-phase synthesis is a method commonly known to those skilled in the art for the synthesis of siRNA. The nucleoside monomers used in the present application are commercially available.

[0079] The siRNA conjugate formed by the GalNAc and the siRNA molecule according to the present application has a structure as shown in Error! Reference source not found..

[0080] It is worth mentioning that the raw materials used in the present application are all ordinary commercially available products, and their sources are not specifically limited.

[0081] The following raw material sources are exemplary:

[0082] The abbreviations for the nucleotide monomers used in the nucleic acid sequence listing are found in Table 1. It is noted that when these monomers are present in an oligonucleotide, they are connected to one another by 5'-3'-phosphodiester linkages; and it is understood that when a nucleotide contains a 2'-fluoro modification, then the fluoro replaces the hydroxyl at that position in the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluoro nucleotide).

[0083] Table 1

[0084] Example 1

[0085] siRNA synthesis

[0086] siRNA was designed against C5 gene, the transcripts used for the design included NM_001735.3, NM_001317163.2, NM_001317164.2, NM_053020.1, XM_015116942.2, XM_015116941.2, XM_028834901.1, NM_010406.3, XM_015436776.1, XM_005580915.2. The siRNAs designed according to the above gene transcripts were synthesized by Suzhou Jimake Gene Co., Ltd., and the specific sequences are shown in the table:

[0087] Table 2

[0088] Example 2

[0089] Inhibition rate of unmodified siRNA on C5 gene

[0090] The present example detects the in-target activity of unmodified siRNA for inhibiting C5 gene, using psiCHECK2 vector to construct plasmid vector for detection, psiCHECK2 vector is a plasmid vector, which can monitor the change of expression of target gene fused with reporter gene, the vector uses Renilla luciferase as the main reporter gene, the target fragment is cloned into the multiple cloning site downstream of the Renilla luciferase translation termination codon, the RNAi process against the target gene triggered by the synthesized siRNA leads to the cleavage and subsequent degradation of the fusion mRNA; by detecting the change of Renilla luciferase activity, it can be determined whether there is a targeting relationship between siRNA and the target gene fragment, the experimental process is as follows:

[0091] 1. Constructing detection plasmid C5-psiCHECK2

[0092] The psiCHECK-2 (Promega TM , item number C8021) plasmid was used to construct the detection plasmid, the detection plasmid contains an insertion sequence, which is designed according to the mRNA sequence shown in Genbank registration number NM_001735.3, synthesized by Suzhou Jimake Gene Co., Ltd., and a single copy of the insertion sequence is cloned into the Xho I / Not I site of the psiCHECK-2 plasmid to obtain the detection plasmid, and the specific plasmid insertion sequence is shown in Table 3:

[0093] Table 3

[0094] 2. Cell culture and transfection

[0095] In a 96-well plate, 5 μL siRNA, 12.5 μL Opti-MEM containing 20 ng C5-psiCHECK2 detection plasmid, 32.5 μL Opti-MEM and 0.3 μL Lipofectamine 2000 (purchased from Invitrogen, item number 11668-019) were added to each well and incubated at room temperature for 15 minutes. Then 50 μL of 1×10 4DMEM complete medium (purchased from Transgen Biotech, Cat. No. FI101-01) for 293T cells was incubated at 37℃ for 24h, and then used for subsequent dual-luciferase assay. The final concentration of siRNA in the experiment was 1 nM.It should be noted that, among them, GRD3408001UM-GRD3408039UM uses C5-psiCHECK2 detection plasmid of GRD342208V01, GRD3408040UM-GRD3408098UM uses C5-psiCHECK2 detection plasmid of GRD342208V02, GRD3408099UM-GRD3408166UM uses C5-psiCHECK2 detection plasmid of GRD342208V03, GRD3408156UM-GRD3408199UM uses C5-psiCHECK2 detection plasmid of GRD342208V04, GRD3408196UM-GRD3408253UM uses C5-psiCHECK2 detection plasmid of GRD342208V05, GRD3408250UM-GRD3408283UM uses C5-psiCHECK2 detection plasmid of GRD342208V06, GRD3408282UM-GRD3408307UM uses C5-psiCHECK2 detection plasmid of GRD342208V07, GRD3408299UM-GRD3408352UM uses C5-psiCHECK2 detection plasmid of GRD342208V08, GRD3408352UM-GRD3408388UM uses C5-psiCHECK2 detection plasmid of GRD342208V09, GRD3408389UM-GRD3408420UM uses C5-psiCHECK2 detection plasmid of GRD342208V10, GRD3408419UM-GRD3408449UM uses C5-psiCHECK2 detection plasmid of GRD342208V11, GRD3408450UM-GRD3408464UM uses C5-psiCHECK2 detection plasmid of GRD342208V12, GRD3408459UM-GRD3408489UM uses C5-psiCHECK2 detection plasmid of GRD342208V13, GRD3408485UM-GRD3408505UM uses C5-psiCHECK2 detection plasmid of GRD342208V14, GRD3408504UM-GRD3408537UM uses C5-psiCHECK2 detection plasmid of GRD342208V15, GRD3408516UM-GRD3408546UM uses C5-psiCHECK2 detection plasmid of GRD342208V16.

[0096] 3. Dual luciferase assay

[0097] The 5x lysis solution in the dual-luciferase assay kit (purchased from Promega, item number E2940) was diluted with water to 1x lysis solution. The cells obtained in step 2 were taken, the supernatant was discarded, PBS buffer (purchased from Hyclone, item number SH30256.01) was used to dilute and wash twice per well, 50 μL / well of 1x lysis solution was added to each cell plate, and the plate was lysed at room temperature for 20 min to obtain a lysed cell plate; 30 μL / well of lysis solution was taken from the lysed cell plate and added to a non-transparent 96-well detection plate, the dual-luciferase assay kit was taken, two substrates were prepared according to the instructions, and the two substrates were added to a new 96-well plate, 30 μL / well of substrate 1 and substrate 2 were added, and the multifunctional enzyme marker was detected after each addition of substrate to obtain the numerical results of firefly luciferase and renilla luciferase, respectively.

[0098] The luminescence ratio of each well of the enzyme-labeled plate was calculated = Renilla / Firefly, and the luminescence ratio of each test group or control group was the average of the luminescence ratios of three culture wells; the luminescence ratio of each test group was normalized based on the luminescence ratio of the control group to obtain the ratio R of luminescence ratio (test) / luminescence ratio (control), which represented the expression level of the Renilla reporter gene, i.e. the relative residual activity. The inhibition rate of siRNA was calculated as (1-R) x 100%.

[0099] The inhibition rate of siRNA is shown in

[0100] Table:

[0101] Table 4

[0102] As can be seen from the table, the unmodified siRNA synthesized by the application has a good inhibitory effect on the expression of the complement component C5 gene, and the inhibition efficiency is almost more than 80%.

[0103] Example 3

[0104] siRNA modification and synthesis

[0105] This embodiment provides a modified siRNA for inhibiting C5, which comprises GRD3408001M1-GRD3408485M1.

[0106] The sense strand of GRD3408001M1-GRD3408546M1 is modified from the sequence shown in SEQ ID NO. 1-SEQ ID NO. 546 respectively, and the nucleotides at position 1 and 2, position 2 and 3 are connected by phosphorothioate group in the direction from 5' end to 3' end, the nucleotides at position 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 are methoxy modified nucleotides, and the nucleotides at position 7, 8, 9 are fluorine modified nucleotides; the antisense strand of GRD3408001M1-GRD3408546M1 is obtained by chemical modification from the sequence selected from SEQ ID NO. 547-SEQ ID NO. 1092, and the nucleotides at position 1 and 2, position 2 and 3, position 19 and 20, position 20 and 21 are connected by phosphorothioate group in the direction from 5' end to 3' end, the nucleotides at position 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 are methoxy modified nucleotides, and the nucleotides at position 2, 6, 14, 16 are fluorine modified nucleotides.

[0107] The modified sequences are named as GRD3408001M1-GRD3408546M1.

[0108] The number M1.1 only represents batch, and is exactly the same as M1 in modification method.

[0109] The RNA is synthesized by phosphoramidite solid phase synthesis according to the type and sequence of nucleotides in the siRNA to be prepared, and the nucleotide monomers used in the synthesis can be purchased.

[0110] Example 4

[0111] Inhibition rate of modified siRNA

[0112] The experimental procedure is the same as that in Example 2, and the results are shown in Table 5:

[0113] Table 5

[0114] As can be seen from the table, the modified partial siRNA constructed by the application has a good inhibitory efficiency on the expression of the complement component C5 gene, and there are 124 siRNAs with an inhibitory rate greater than 90% at a concentration of 1 nM; and there are 5 siRNAs with an inhibitory rate greater than 90% at a concentration of 0.1 nM.

[0115] Example 5

[0116] Animal experiment:

[0117] In an embodiment of the application, the siRNA conjugate formed by the GalNAc and the siRNA molecule has the structure shown in the figure.

[0118] 1. Synthesis of siRNA used in animal experiments Alnylam pharmaceuticals, Inc. reported that siRNA based on GalNAc conjugation technology has interference activity in mice in vivo (Nair et al., J. Am. Chem. Soc., 2014, 136, 16958-16961), and the literature reported that siRNA conjugated to three clusters of GalNAc exhibited good delivery activity in in vivo and in vitro experiments, referring to the preparation method in the above-mentioned literature, GalNAc conjugated siRNA shown in the example was used to obtain GalNAc conjugated siRNA, and the siRNA is shown in the table:

[0119] 2. GRD3408001M1G-GRD3408485M1G represents that the 3' end of the sense strand of the modified sequence GRD3408001M1-GRD3408485M1 is coupled with GalNAc on the basis of GRD3408001M1-GRD3408485M1.

[0120] 3.The sense strand of GRD3408001M3-GRD3408485M3 is modified from the sequence set forth in SEQ ID NO. 1-SEQ ID NO. 546, respectively, in the direction from 5’ end to 3’ end, the nucleotides at position 1 and 2, position 2 and 3 are connected by phosphorothioate linkage, the nucleotides at position 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 are methoxy modified nucleotides, the nucleotides at position 5, 7, 8, 9 are fluorine modified nucleotides; the antisense strand of GRD3408001M3-GRD3408485M3 is obtained by chemical modification from the sequence selected from the group consisting of SEQ ID NO. 547-SEQ ID NO. 1092, in the direction from 5’ end to 3’ end, the nucleotides at position 1 and 2, position 2 and 3, position 19 and 20, position 20 and 21 are connected by phosphorothioate linkage, the nucleotides at position 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 are methoxy modified nucleotides, the nucleotides at position 2, 6, 14, 16 are fluorine modified nucleotides.GRD3408001M3GVP-GRD3408485M3GVP indicates that on the basis of the modified sequence GRD3408001M3-GRD3408485M3, the 3’ end of the sense strand of GRD3408001M3-GRD3408485M3 is coupled with GalNAc, and the nucleotide at position 1 of the 5’ end of the antisense strand is a VP modified nucleotide.

[0121] 4.The sense strand of GRD3408001M21-GRD3408485M21 is modified from the sequence set forth in SEQ ID NO. 1-SEQ ID NO. 546, respectively, in the direction from 5’ end to 3’ end, the nucleotides at position 1 and 2, and the nucleotides at position 2 and 3 are connected through phosphorothioate linkage, the nucleotides at position 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy modified nucleotides, and the nucleotides at position 7, 8, and 9 are fluoro modified nucleotides; the antisense strand of GRD3408001M21-GRD3408485M21 is obtained by chemically modifying the sequence selected from the group consisting of SEQ ID NO. 547-SEQ ID NO. 1092, in the direction from 5’ end to 3’ end, the nucleotides at position 1 and 2, the nucleotides at position 2 and 3, the nucleotides at position 19 and 20, and the nucleotides at position 20 and 21 are connected through phosphorothioate linkage, the nucleotides at position 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy modified nucleotides, the nucleotide at position 2 is a deoxynucleotide, and the nucleotides at position 6, 14, and 16 are fluoro modified nucleotides.GRD3408001M21GVP-GRD3408485M21GVP indicates that on the basis of the modified sequence GRD3408001M21-GRD3408485M21, the 3’ end of the sense strand of GRD3408001M21-GRD3408485M21 is coupled with GalNAc, and the nucleotide at position 1 of the 5’ end of the antisense strand is a VP modified nucleotide.

[0122] 5.The sense strand of GRD3408001M22-GRD3408485M22 is modified from the sequence set forth in SEQ ID NO. 1-SEQ ID NO. 546, respectively, in the direction from 5’ end to 3’ end, the nucleotides at position 1 and 2, and the nucleotides at position 2 and 3 are connected through phosphorothioate linkage, the nucleotides at position 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 are methoxy modified nucleotides, and the nucleotides at position 7, 8, and 9 are fluorine modified nucleotides; the antisense strand of GRD3408001M22-GRD3408485M22 is obtained by chemically modifying the sequence selected from the group consisting of SEQ ID NO. 547-SEQ ID NO. 1092, in the direction from 5’ end to 3’ end, the nucleotides at position 1 and 2, the nucleotides at position 2 and 3, the nucleotides at position 19 and 20, and the nucleotides at position 20 and 21 are connected through phosphorothioate linkage, the nucleotides at position 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are methoxy modified nucleotides, the nucleotide at position 6 is a deoxynucleotide, and the nucleotides at position 2, 14, and 16 are fluorine modified nucleotides.GRD3408001M22GVP-GRD3408485M22GVP indicates that on the basis of the modified sequence GRD3408001M22-GRD3408485M22, the 3’ end of the sense strand of GRD3408001M22-GRD3408485M22 is coupled with GalNAc, and the nucleotide at position 1 of the 5’ end of the antisense strand is a VP modified nucleotide.

[0123] 6.The sense strand of GRD3408001M27-GRD3408485M27 is modified from the sequence shown in SEQ ID NO.1-SEQ ID NO.546, respectively, and the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions are connected by a phosphorothioate group in the direction from the 5' end to the 3' end, the nucleotides at the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are methoxy modified nucleotides, and the nucleotides at the 5th, 7th, 8th, 9th positions are fluorine modified nucleotides; the antisense strand of GRD3408001M27-GRD3408485M27 is obtained by chemical modification of the sequence selected from SEQ ID NO.547-SEQ ID NO.1092, respectively, and the nucleotides at the 1st and 2nd positions, the 2nd and 3rd positions, the 19th and 20th positions, the 20th and 21st positions are connected by a phosphorothioate group in the direction from the 5' end to the 3' end, the nucleotides at the 1st, 3rd, 4th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions are methoxy modified nucleotides, the nucleotide at the 5th position is a deoxyribonucleotide, and the nucleotides at the 2nd, 6th, 14th, 16th positions are fluorine modified nucleotides.GRD3408001M27GVP-GRD3408485M27GVP indicates that on the basis of the modified sequence GRD3408001M27-GRD3408485M27, the 3' end of the sense strand of GRD3408001M27-GRD3408485M27 is coupled with GalNAc, and the nucleotide at the 1st position of the 5' end of the antisense strand is a VP modified nucleotide.

[0124] The.1 and.2 in the table numbers G.1 and G.2 only represent batches, and the modification methods are exactly the same.

[0125] Table 6

[0126] 2.In each group of mice, GalNAc conjugated siRNA or saline control is administered subcutaneously once, 100 μL is injected subcutaneously on the nape, the mice are sacrificed on a certain day after administration, the liver samples are collected and quickly frozen in liquid nitrogen, the liver mRNA is extracted and analyzed by RT-qPCR method, and the detection steps of RT-qPCR are as follows:

[0127] (1) RNA extraction:

[0128] 1) Take 20 mg of mouse liver tissue, add 1 mL of Trizol Lysis Buffer (purchased from Life technology, item number 410701) to grind and lyse the tissue, and transfer the completely dissolved mixture to an RNase-free 1.5 mL centrifuge tube; shake vigorously for about 15 s to fully lyse the tissue cells, and stand at room temperature (25°C) for 5 min;

[0129] 2) Carefully open the tube cap, add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., item number 20140925); shake vigorously for 20 s, stand at room temperature (25°C) for 3 min; centrifuge at 4°C, 12000 x g for 20 min;

[0130] 3) After centrifugation, carefully take the centrifuge tube to the centrifuge tube rack, and transfer the supernatant water phase to a new 2.0 mL centrifuge tube, and add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., item number 20210802) to the supernatant water phase, and mix well by inverting;

[0131] 4) Take a purification column with a collection tube (purchased from VWI company, item number 11822AG0627), and add 700 μL of the mixture in step 3) to it, and stand for 2 min; centrifuge at 4°C, 10000 x g for 1 min, and discard the filtrate; repeat the above steps with the remaining mixture;

[0132] 5) Add 700 μL of 80% (v / v) ethanol to the purification column, centrifuge at 4°C, 10000 x g for 1 min, and discard the filtrate;

[0133] 6) Add 700 μL of 80% (v / v) ethanol to the purification column, centrifuge at 4°C, 10000 x g for 1 min, and discard the filtrate;

[0134] 7) Centrifuge the purification column at 4°C, 10000 x g for 2 min;

[0135] 8) After centrifugation, carefully take out the purification column with a collection tube (if there is liquid in the collection tube, be careful not to splash the liquid onto the purification column), discard the collection tube, and place the purification column in a new 1.5 mL centrifuge tube, add 100 μL of DEPC water to the purification column, and stand at room temperature (25°C) for 2 min; centrifuge at 4°C, 10000 x g for 1 min;

[0136] 9) Collect the RNA solution in step 8) for subsequent experiments;

[0137] (2) RNA reverse transcription

[0138] The experimental steps refer to the product manual of HiScript III RT SuperMix for qPCR (purchased from Novoprotein, with the product number R323-01); the reverse transcription reaction system 20 μL was prepared according to the reverse transcription operation steps in the kit manual, and the total RNA of the cells was reverse transcribed; the reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 37 °C for 15 min, and then at 85 °C for 5 s, 80 μL of DEPC water was added to each reverse transcription reaction system to obtain a cDNA-containing solution;

[0139] (3) qPCR reaction system configuration

[0140] For each reverse transcription reaction system, 4 μL of the above cDNA-containing solution was taken as a template, and the reagents provided by the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, with the product number Q511-02) were used to configure a qPCR reaction system 20 μL on an ice box according to Table 7, wherein Primer 1 and Primer 2 were PCR primer sequences for amplifying the target gene C5 and the internal reference gene GAPDH (as shown in Table 8), respectively; each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and a three-step amplification was performed, with the amplification program being 95 °C pre-denaturation for 10 min, then 95 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 30 s, and repeating the above denaturation, annealing and extension processes for 40 times, to obtain a product W containing the amplified target gene C5 and internal reference gene GAPDH; the product W was then sequentially incubated at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s, and the real-time fluorescence quantitative PCR instrument collected the melting curves of the target gene C5 and the internal reference gene GAPDH in the product W, to obtain the Ct values of the target gene C5 and the internal reference gene GAPDH. The comparative Ct (ΔΔCt) method was used to calculate the relative quantification of the target gene C5 in each test group, and the calculation method was as follows: ΔCt (test group) = Ct (test group target gene) - Ct (test group internal reference gene) ΔCt (control group) = Ct (control group target gene) - Ct (control group internal reference gene) ΔΔCt (test group) = ΔCt (test group) - ΔCt (control group average) ΔΔCt (control group) = ΔCt (control group) - ΔCt (control group average)

[0141] Wherein, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) of the four samples in the control group; thus, each sample in the test group and the control group corresponds to a ΔΔCt value.

[0142] The expression level of C5 mRNA of the test group was normalized based on the control group, and the expression level of C5 mRNA of the control group was defined as 100%.

[0143] Relative expression level of C5 mRNA of the test group = 2^(-ΔΔCt(test group)) x 100%

[0144] Inhibition rate of C5 mRNA of the test group = 1 - relative expression level of C5 mRNA of the test group

[0145] The C5 mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the average value of the saline control group, and the data was expressed as a percentage relative to the saline control group.

[0146] Table 7 DNA amplification reaction system

[0147] Table 8 primer information

[0148] The first animal experiment conditions are shown in Table 9:

[0149] Table 9

[0150] The experimental results are shown in Table 1.

[0151] The second animal experiment conditions are shown in Table 10:

[0152] Table 10

[0153] The experimental results are shown in Table 1.

[0154] The third animal experiment conditions are shown in Table 11, and the experimental results are shown in Figure 4.

[0155] Table 11

[0156] The fourth animal experiment conditions are shown in Table 12, and the experimental results are shown in Table 1.

[0157] Table 12

[0158] The fifth animal experiment conditions are shown in Table 13, and the experimental results are shown in Table 1.

[0159] Table 13

[0160] From the animal experiment results, it can be seen that the modified siRNA synthesized in the application still has a good inhibitory effect on the expression of the complement component C5 gene in the in vivo experiment.

[0161] 3. Each group of mice was subcutaneously administered with GalNAc conjugated siRNA or saline control once, 100 μL was subcutaneously injected in the nape of the neck, serum was taken one day before administration, serum samples were collected every seven days after administration, the expression level of C5 protein in the serum was detected by using an ELISA kit (Thermo Fisher, BMS2088), and the detection method is described in the kit instructions.

[0162] The animal experiment conditions are shown in Table 14:

[0163] Table 14

[0164] The standardization method of C5 protein level is to divide the C5 protein level of each animal at a time point by the expression level of the animal before treatment (in this case, on Day-1) to determine the expression ratio “standardized to pre-treatment”. Then the expression at a specific time point is standardized to the saline control group by dividing the “standardized to pre-treatment” ratio of individual animals by the average “standardized to pre-treatment” ratio of all mice in the saline control group, which enables the expression at each time point to be standardized to the control group, and the specific results are shown in Table 15.

[0165] Table 15

[0166] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. An siRNA for inhibiting the expression of complement component C5, characterized in that, The siRNA comprises a sense strand and an antisense strand at least partially forming a double-stranded region, the sense strand containing at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleic acid sequences shown as any one of SEQ ID NO. 1-SEQ ID NO. 546; the antisense strand containing at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleic acid sequences shown as any one of SEQ ID NO. 547-SEQ ID NO. 1092.

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

3. The siRNA of claim 2, wherein The modified nucleotide is selected from a deoxyribonucleotide, a methoxy-modified nucleotide, a fluoro-modified nucleotide, or a locked nucleic acid-modified nucleotide.

4. The siRNA of claim 3, wherein The methoxy-modified nucleotide is located in the nucleotide sequences of the antisense strand and the sense strand, and, in the direction from the 5' end to the 3' end, at least the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th position of the nucleotide of the sense strand is a methoxy-modified nucleotide, and at least the 1st, 3rd, 4th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st position of the nucleotide of the antisense strand is a methoxy-modified nucleotide; Alternatively, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th position of the nucleotide of the sense strand is a methoxy-modified nucleotide, and at least the 1st, 3rd, 4th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st position of the nucleotide of the antisense strand is a methoxy-modified nucleotide; Alternatively, at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th position of the nucleotide of the sense strand is a methoxy-modified nucleotide, and at least the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st position of the nucleotide of the antisense strand is a methoxy-modified nucleotide.

5. The siRNA of claim 3, wherein The fluoro-modified nucleotide is located in the nucleotide sequences of the antisense strand and the sense strand, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide; Alternatively, at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 6th, 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide; Alternatively, at least the 7th, 8th, 9th position of the nucleotide of the sense strand is a fluoro-modified nucleotide, and at least the 2nd, 6th, 14th, 16th position of the nucleotide of the antisense strand is a fluoro-modified nucleotide; Alternatively, at least the 5th, 7th, 8th, 9th nucleotides of the sense strand are fluorine-modified nucleotides, and at least the 2nd, 6th, 14th, 16th nucleotides of the antisense strand are fluorine-modified nucleotides.

6. The siRNA of claim 3, wherein At least one of the phosphates in the phospho-sugar backbone of at least one of the single strands of the siRNA is a phosphorothioate, and, in the 5' end to 3' end direction, at least the 1st and 2nd, 2nd and 3rd nucleotides of the sense strand are linked by phosphorothioate, and at least the 1st and 2nd, 2nd and 3rd, 19th and 20th, 20th and 21st nucleotides of the antisense strand are linked by phosphorothioate.

7. The siRNA of claim 3, wherein The deoxyribonucleotides are located in the antisense strand of the nucleotide sequence, and, in the 5' end to 3' end direction, at least one of the 2nd, 5th, 6th nucleotides of the antisense strand is a deoxyribonucleotide.

8. The siRNA of claim 3, wherein In the 5' end to 3' end direction, the 1st and 2nd, 2nd and 3rd nucleotides of the sense strand are linked by phosphorothioate, the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th nucleotides are methoxy-modified nucleotides, and the 7th, 8th, 9th nucleotides are fluorine-modified nucleotides; the 1st and 2nd, 2nd and 3rd, 19th and 20th, 20th and 21st nucleotides of the antisense strand are linked by phosphorothioate, the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st nucleotides are methoxy-modified nucleotides, and the 2nd, 6th, 14th, 16th nucleotides are fluorine-modified nucleotides. In the 5' end to 3' end direction, the 1st and 2nd, 2nd and 3rd nucleotides of the sense strand are linked by phosphorothioate, the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th nucleotides are methoxy-modified nucleotides, and the 5th, 7th, 8th, 9th nucleotides are fluorine-modified nucleotides; the 1st and 2nd, 2nd and 3rd, 19th and 20th, 20th and 21st nucleotides of the antisense strand are linked by phosphorothioate, the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st nucleotides are methoxy-modified nucleotides, and the 2nd, 6th, 14th, 16th nucleotides are fluorine-modified nucleotides. or the nucleotides at the 1st and 2nd, 2nd and 3rd positions of the sense strand are connected by phosphorothioate group, the nucleotides at the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are methoxy modified nucleotides, and the nucleotides at the 7th, 8th, 9th positions are fluoro modified nucleotides; the nucleotides at the 1st and 2nd, 2nd and 3rd, 19th and 20th, 20th and 21st positions of the antisense strand are connected by phosphorothioate group, the nucleotides at the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions are methoxy modified nucleotides, the nucleotide at the 2nd position is a deoxynucleotide, and the nucleotides at the 6th, 14th, 16th positions are fluoro modified nucleotides; or the nucleotides at the 1st and 2nd, 2nd and 3rd positions of the sense strand are connected by phosphorothioate group, the nucleotides at the 1st, 2nd, 3rd, 4th, 5th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are methoxy modified nucleotides, and the nucleotides at the 7th, 8th, 9th positions are fluoro modified nucleotides; the nucleotides at the 1st and 2nd, 2nd and 3rd, 19th and 20th, 20th and 21st positions of the antisense strand are connected by phosphorothioate group, the nucleotides at the 1st, 3rd, 4th, 5th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions are methoxy modified nucleotides, the nucleotide at the 6th position is a deoxynucleotide, and the nucleotides at the 2nd, 14th, 16th positions are fluoro modified nucleotides; or the nucleotides at the 1st and 2nd, 2nd and 3rd positions of the sense strand are connected by phosphorothioate group, the nucleotides at the 1st, 2nd, 3rd, 4th, 6th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th positions are methoxy modified nucleotides, and the nucleotides at the 5th, 7th, 8th, 9th positions are fluoro modified nucleotides; the nucleotides at the 1st and 2nd, 2nd and 3rd, 19th and 20th, 20th and 21st positions of the antisense strand are connected by phosphorothioate group, the nucleotides at the 1st, 3rd, 4th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 15th, 17th, 18th, 19th, 20th, 21st positions are methoxy modified nucleotides, the nucleotide at the 5th position is a deoxynucleotide, and the nucleotides at the 2nd, 6th, 14th, 16th positions are fluoro modified nucleotides.

9. The siRNA according to any one of claims 1 to 8, wherein The 5' end of the antisense strand of the siRNA comprises at least one modification.

10. The siRNA of claim 9, wherein The 5' end of the antisense strand of the siRNA comprises at least one 5'-(E)-vinyl phosphonate modification.

11. The siRNA according to any one of claims 1 to 8, wherein The 3' end of the sense strand of the siRNA is coupled with a ligand.

12. The siRNA of claim 11, wherein The ligand is GalNAc.

13. Use of the siRNA of any one of claims 1-12 in the preparation of a C5 complement targeting inhibitor.

14. Use according to claim 13, characterized in that, The C5 complement targeting inhibitor is used for treating a disease caused by complement abnormality or for improving complement abnormality caused by a disease. The C5 complement targeting inhibitor is used for treating a disease caused by complement abnormality or for improving complement abnormality caused by a disease.

15. Use of the siRNA according to any one of claims 1 to 12 for the manufacture of a medicament.

16. A medicament, characterized by comprising: comprising the siRNA according to any one of claims 1 to 12.

17. A treatment method, characterized in that, comprising administration of the siRNA according to any one of claims 1 to 12.

18. A genetically engineered article of manufacture, comprising, comprising, expressing or secreting the siRNA according to any one of claims 1 to 12.

19. The genetically engineered article of manufacture of claim 18, wherein, selected from the group consisting of an expression vector or a genetically engineered cell.

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