CN117947024A

CN117947024A - SiRNA sequence for effectively inhibiting expression of complement C3 factor and application thereof

Info

Publication number: CN117947024A
Application number: CN202311392216.2A
Authority: CN
Inventors: 贾宏丽; 林美娜; 杨脉; 陈晓萌; 赵宣
Original assignee: Jenkem Technology Co Ltd
Current assignee: Jenkem Technology Co Ltd
Priority date: 2022-10-27
Filing date: 2023-10-25
Publication date: 2024-04-30

Abstract

The invention provides an interfering RNA for inhibiting expression of complement C3 factor and application thereof. The interfering RNA is siRNA, and the siRNA comprises SEQ ID NO:1-36, which is delivered into a cell of interest, which is effective in inhibiting the expression of complement C3 factor, can be used for studying the regulation of complement activation pathway, and is of great value for the preparation of a medicament for the treatment of a disease characterized by an increase in the expression of complement C3 factor.

Description

SiRNA sequence for effectively inhibiting expression of complement C3 factor and application thereof

Technical Field

The invention relates to the technical fields of molecular biology and biological medicine, in particular to siRNA for effectively inhibiting complement C3 factor gene expression.

Background

The complement system is a group of soluble proteins found in human or vertebrate serum and body fluids, and a group of membrane-bound proteins and complement receptors found on the surfaces of blood cells and other tissue cells. Under physiological conditions, most of complement intrinsic components exist in zymogen or non-activated forms, and after being activated by certain substances, the complement intrinsic components can be triggered to generate enzymatic cascade reactions according to a certain sequence, so as to generate various substances with biological activity, assist the defense mechanism against infection and mediate various inflammatory processes, and cause a plurality of immunological biological effects such as tissue injury, inflammatory reaction, allergic reaction and the like. The complement system plays an important role in maintaining the balance of the environment in the body and is an important component of the innate immunity of the body. When complement is overactivated, inflammatory reactions can be induced and affect the coagulation and fibrinolytic system, causing pathological damage.

The complement activation pathway can be broadly divided into three, the classical pathway (first pathway), the alternative pathway (second pathway) and the lectin pathway. All three activation pathways need to participate via C3 to eventually form the membrane attack complex and act, so activation of C3 is the most critical loop in the complement system.

C3 is the complement component with the greatest content in blood. Under normal conditions, C3 can be naturally and slowly cleaved, and small amounts of C3b produced can be rapidly inactivated by factor I; when C3 is activated, the C3B cleavage product can be synthesized into new C3bBb under the participation of the factor B and the factor D, the new C3bBb is further cleaved by the latter, and the positive feedback effect greatly amplifies the activation and forms inflammatory factor storm when serious.

In general, complement C3 elevation is seen in certain acute inflammatory or infectious disease early stages. In recent years, studies have found that C3 levels are also elevated in senile macular degeneration (AMD) and some tumorigenesis, with significant implications for morbidity.

1. Complement factor C3 and age-related macular degeneration

Age-related macular degeneration is a progressive retinal disorder characterized by drusen formation, changes in pigment in the macular area, geographic atrophy, and choroidal neovascularization with exudation, with over 3300 thousands of patients worldwide. In the choroidal tissues of patients with age-related macular degeneration, there is inflammatory cell infiltration, and various immunologically active substances appear in drusen, and complement activating molecules are significantly elevated. In the retinal pigment epithelium substructure, drusen and choroid, where age-related macular degeneration occurs, the lytic component of complement factor C3 occurs. Studies have shown that C3a can accelerate the formation of a model of vascular endothelial growth factor treated neovascular age-related macular degeneration; when the genetic technology is adopted to inhibit the receptor expression of C3a, the vascular endothelial growth factor expression, lymphocyte recruitment and neovascularization after laser injury are reduced; the use of antibodies to neutralize C3a or to block its receptor with drugs may also reduce neovascularization. Complement factor C3 is thus a potentially sound candidate target.

Further studies have shown that the variation of the complement factor C3 gene is significantly associated with age-related macular degeneration, suggesting that the complement pathway may play an important role in the etiology of age-related macular degeneration. As identified, variations in Lys155Gln, lys65Gln, arg735Trp, ser1619Arg, arg80Gly, etc., may result in loss of genetics, leading to increased susceptibility of an individual to age-related macular degeneration.

Although the complement factor C3 genotype had a strong linkage disequilibrium between Arg102Gly and Leu314Pro, making it difficult to distinguish whether it acted on disease occurrence, the data showed that Arg102Gly polymorphisms were distributed in age-related macular degeneration patients quite differently from the control group (p=0.001), the GC genotype had an OR of 1.69 (1.15-2.49) and the GG genotype had an OR of 6.48 (1.87-22.43) compared to the CC genotype. The G allele has an OR of 2.31 (0.48-11) compared to the C allele. The GG and GC genotypes and the G allele are significantly associated with both progressive age-related macular degeneration, and individuals carrying the GG genotype are 6-fold more at risk for developing age-related macular degeneration than individuals carrying the CC genotype, and are a relatively strong susceptibility gene for progressive age-related macular degeneration (exudative and geographic atrophy) complement factor C3.

In view of the above studies, small molecule drugs Pegcetacoplan specifically targeted to complement factor C3 were developed for geographic atrophy.

2. Complement factor C3 and tumors

Complement factor C3 gene is closely related to the occurrence and the prognosis of various tumor diseases such as lung cancer, gastric cancer, ovarian cancer, skin cancer and the like.

Tumor microenvironments play a critical role in tumorigenesis, progression, metastasis and recurrence. Studies demonstrate that complement activation has an immunomodulatory effect in the tumor microenvironment. Complement can exert two distinct effects on tumor cells: anti-tumor effects, manifested by membrane-bound complement regulatory proteins, low expression of cell membrane-lytic attack complexes and moderate C5a concentrations; the pro-tumor effect is expressed as high expression and high concentration of C5a by membrane-bound complement regulatory proteins, sub-lytic membrane attack complexes.

Imbalance in complement activation and deposition of complement proteins are found in many types of tumors. In individuals with lung cancer and gastric cancer, both the mRNA level of C3a and the average protein level were significantly increased. When complement factors C3, C5a or their receptors or membrane bound complement regulatory proteins are blocked or silenced, tumor migration, proliferation is inhibited, and disease progression and survival of patients are improved. In complement factor C3 deficient mice, both primary lung cancer and metastatic cancer are strongly inhibited, even though no tumor presence is detectable in many individuals. This growth inhibition was associated with an increase in ifnγ ⁺/TNFα⁺/IL10⁺CD4⁺ and CD8 ⁺ T cell numbers. In various models of in situ inoculation of tumor cell lines, complement factor C3/C3 receptor autocrine signaling loops were found to be involved in the regulation of tumor growth. These evidences all indicate that complement activation is an important regulatory factor in the progression of lung cancer, driving immune escape by complement factor C3-dependent means. These results demonstrate that complement factor C3 plays a role in tumors, and can be used as a biomarker and a potential therapeutic target for such metastatic tumors.

In patent US20080090997A1, sequences 557 and 549 are identical to sequences 6 and 9, respectively, of this patent, but retrieved, the corresponding sequences in the description of this patent are both TRPV1 (nm_080704) targeting, which is not the C3 mRNA sequence.

Disclosure of Invention

The invention designs specific siRNA aiming at mRNA of complement C3 factor, and delivers the mRNA into target cells, thereby realizing the inhibition of expression of complement C3 factor, and providing a new thought for confirming that the increase of expression of C3 factor is a pathogenic factor or the treatment of diseases involving in the factor.

In order to detect the inhibition effect of the siRNA corresponding to the complement C3 factor, 16 siRNAs are prepared aiming at the C3 factor. In a cell experiment, firstly, the prepared siRNA is delivered into cultured cells, and cell samples are collected and RNA is extracted at a preset time after transfection; after reverse transcription of RNA into cDNA, ct value was obtained by a real-time quantitative PCR method. And (3) carrying out normalization treatment on each group of data to obtain the relative expression condition of the C3mRNA, so as to compare the inhibition effect of each siRNA on the expression of the C3 mRNA.

In a first aspect of the invention, there is provided an interfering RNA targeting the complement C3 factor encoding gene.

Preferably, the interfering RNA inhibits expression of complement C3 factor.

Preferably, the interfering RNA is obtained by any means known in the art, such as chemical synthesis, in vitro transcription, enzymatic digestion or in vivo transcription.

In one embodiment of the invention, the interfering RNA is chemically synthesized.

Preferably, the interfering RNA comprises SEQ ID NO:1-36 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36).

Preferably, the interfering RNA consists of SEQ ID NO:1-36 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36).

In one embodiment of the invention, the interfering RNA comprises the sequence of SEQ ID NO: 1. 2,3, 4, 5, 6, 8, 9, 11, 12, 13, 15, 16, 17, 19, 20, 21, 22, 23, 24, 26, 27, 29, 30, 31, 33, 34, or 35 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28).

In one embodiment of the invention, the interfering RNA consists of the sequence set forth in SEQ ID NO: 1. 2,3,4, 5, 6, 8, 9, 11, 12, 13, 15, 16, 17, 19, 20, 21, 22, 23, 24, 26, 27, 29, 30, 31, 33, 34, or 35 (e.g., 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28). In one embodiment of the invention, the interfering RNA comprises the sequence of SEQ ID NO: 5. 6, 8, 9, 11, 12, 13, 15, 16, 23, 24, 26, 27, 29, 30, 31, 33, or 34 (e.g., 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18).

In one embodiment of the invention, the interfering RNA consists of the sequence set forth in SEQ ID NO: 5.6, 8, 9, 11, 12, 13, 15, 16, 23, 24, 26, 27, 29, 30, 31, 33, or 34 (e.g., 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18).

In one embodiment of the invention, the interfering RNA comprises the sequence of SEQ ID NO: 6. 9, 11, 12, 15, 16, 24, 27, 29, 30, 33, or 34 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

In one embodiment of the invention, the interfering RNA consists of the sequence set forth in SEQ ID NO: 6. 9, 11, 12, 15, 16, 24, 27, 29, 30, 33, or 34 (e.g., 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12).

In one embodiment of the invention, the interfering RNA comprises the sequence of SEQ ID NO: 11. 12, 16, 29, 30 or 34 (e.g., 1, 2, 3, 4, 5 or 6).

In one embodiment of the invention, the interfering RNA consists of the sequence set forth in SEQ ID NO: 11. 12, 16, 29, 30 or 34 (e.g., 1, 2, 3, 4, 5 or 6).

In one embodiment of the invention, the interfering RNA comprises the sequence of SEQ ID NO: 12. 16, 30 or 34 (e.g., 1, 2,3 or 4).

In one embodiment of the invention, the interfering RNA consists of the sequence set forth in SEQ ID NO: 12. 16, 30 or 34 (e.g., 1, 2, 3 or 4).

Preferably, the interfering RNA comprises a sense strand and/or an antisense strand.

Preferably, the interfering RNA comprises a sense strand and an antisense strand.

Further preferably, the sense strand and the antisense strand are complementarily paired.

Preferably, the sense strand of the interfering RNA comprises SEQ ID NO:1-18 (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18).

Preferably, the antisense strand of the interfering RNA comprises SEQ ID NO:19-36 (e.g., 1,2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18).

Preferably, the sense strand of the interfering RNA is as shown in SEQ ID NO:1-18 (e.g., 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18).

Preferably, the antisense strand of the interfering RNA is as shown in SEQ ID NO:19-36 (e.g., 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18).

In one embodiment of this aspect, the sense strand of the interfering RNA comprises SEQ ID NO: 1.2, 3, 4, 5, 6, 8, 9, 11, 12, 13, 15, 16, or 17, or two or more (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14).

In one embodiment of this aspect, the antisense strand of the interfering RNA comprises the sequence of SEQ ID NO: 19. 20, 21, 22, 23, 24, 26, 27, 29, 30, 31, 33, 34, or 35, or two or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14).

In one specific embodiment of this aspect, the sense strand of the interfering RNA is as set forth in SEQ ID NO: 1.2, 3, 4, 5, 6, 8, 9, 11, 12, 13, 15, 16, or 17 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14).

In one embodiment of this aspect, the antisense strand of the interfering RNA is as set forth in SEQ ID NO: 19. 20, 21, 22, 23, 24, 26, 27, 29, 30, 31, 33, 34, or 35 (e.g., 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14).

In one embodiment of the invention, the sense strand of the interfering RNA comprises the sequence of SEQ ID NO: 5. 6, 8, 9, 11, 12, 13, 15 or 16 (e.g., 1,2, 3, 4, 5, 6, 7, 8 or 9).

In one embodiment of the invention, the antisense strand of the interfering RNA comprises the sequence of SEQ ID NO: 23. 24, 26, 27, 29, 30, 31, 33, or 34 (e.g., 1,2, 3,4, 5, 6, 7, 8, or 9).

In one embodiment of the invention, the sense strand of the interfering RNA is as set forth in SEQ ID NO: 5. 6, 8, 9, 11, 12, 13, 15, or 16 (e.g., 1,2,3,4, 5,6,7, 8, or 9).

In one embodiment of the invention, the antisense strand of the interfering RNA is as shown in SEQ ID NO: 23. 24, 26, 27, 29, 30, 31, 33, or 34 (e.g., 1,2,3, 4, 5, 6, 7, 8, or 9).

In one embodiment of the invention, the sense strand of the interfering RNA comprises the sequence of SEQ ID NO: 6. 9, 11, 12, 15 or 16 (e.g., 1,2,3, 4,5 or 6).

In one embodiment of the invention, the antisense strand of the interfering RNA comprises the sequence of SEQ ID NO: 24. 27, 29, 30, 33 or 34 (e.g., 1,2, 3, 4, 5 or 6).

In one embodiment of the invention, the sense strand of the interfering RNA is as set forth in SEQ ID NO: 6. 9, 11, 12, 15, 16 (e.g., 1, 2, 3, 4, 5, or 6).

In one embodiment of the invention, the antisense strand of the interfering RNA is as shown in SEQ ID NO: 24. 27, 29, 30, 33 or 34 (e.g., 1,2, 3, 4, 5 or 6).

In one embodiment of the invention, the sense strand of the interfering RNA comprises the sequence of SEQ ID NO: 11. 12 or 16 (e.g., 1,2 or 3).

In one embodiment of the invention, the antisense strand of the interfering RNA comprises the sequence of SEQ ID NO: 29. 30 or 34 (e.g., 1,2 or 3).

In one embodiment of the invention, the sense strand of the interfering RNA is as set forth in SEQ ID NO: 11. 12 or 16 (e.g., 1,2, or 3).

In one embodiment of the invention, the antisense strand of the interfering RNA is as shown in SEQ ID NO: 29. 30 or 34 (e.g., 1,2, or 3).

In one embodiment of the invention, the sense strand of the interfering RNA comprises the sequence of SEQ ID NO:12 or 16, or both.

In one embodiment of the invention, the antisense strand of the interfering RNA comprises the sequence of SEQ ID NO:30 or 34 or a nucleotide sequence set forth in any one or both of seq id no.

In one embodiment of the invention, the sense strand of the interfering RNA is as set forth in SEQ ID NO:12 or 16, or both.

In one embodiment of the invention, the antisense strand of the interfering RNA is as shown in SEQ ID NO:30 or 34, or both.

According to the specific embodiment needs, the interference RNA is composed of sense strand and antisense strand pairing, for example:

(A) The interfering RNA consists of a sequence containing SEQ ID NO:1 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:1, and a sense strand comprising SEQ ID NO:19 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:19, and an antisense strand shown in FIG. 19.

(B) The interfering RNA consists of a sequence containing SEQ ID NO:2 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:2, and a sense strand comprising SEQ ID NO:20 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:20, and an antisense strand shown in seq id no.

(C) The interfering RNA consists of a sequence containing SEQ ID NO:3 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:3, and a sense strand comprising SEQ ID NO:21 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:21, and an antisense strand shown in seq id no.

(D) The interfering RNA consists of a sequence containing SEQ ID NO:4 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:4, and a sense strand comprising SEQ ID NO:22 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:22, and an antisense strand shown in seq id no.

(E) The interfering RNA consists of a sequence containing SEQ ID NO:5 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:5, and a sense strand comprising SEQ ID NO:23 or an antisense strand of the nucleotide sequence shown as SEQ ID NO: 23.

(F) The interfering RNA consists of a sequence containing SEQ ID NO:6 or a sense strand of the nucleotide sequence shown as SEQ ID NO:6, and, a sense strand comprising SEQ ID NO:24 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:24, and an antisense strand shown in seq id no.

(G) The interfering RNA consists of a sequence containing SEQ ID NO:7 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:7, and, a sense strand comprising SEQ ID NO:25 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:25, and an antisense strand shown in seq id no.

(H) The interfering RNA consists of a sequence containing SEQ ID NO:8 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:8, and a sense strand comprising SEQ ID NO:26 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:26, and an antisense strand shown in seq id no.

(I) The interfering RNA consists of a sequence containing SEQ ID NO:9 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:9, and a sense strand comprising SEQ ID NO:27 or an antisense strand of the nucleotide sequence shown as SEQ ID NO: 27.

(J) The interfering RNA consists of a sequence containing SEQ ID NO:10 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:10, and a sense strand comprising SEQ ID NO:28 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:28, and an antisense strand shown in seq id no.

(K) The interfering RNA consists of a sequence containing SEQ ID NO:11 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:11, and a sense strand comprising SEQ ID NO:29 or the antisense strand of the nucleotide sequence shown as SEQ ID NO: 29.

The interfering RNA of (L) consists of a sequence comprising SEQ ID NO:12 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:12, and a sense strand comprising SEQ ID NO:30 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:30, and an antisense strand shown in seq id no.

The interfering RNA of (M) consists of a sequence comprising SEQ ID NO:13 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:13, and a sense strand comprising SEQ ID NO:31 or the antisense strand of the nucleotide sequence shown as SEQ ID NO: 31.

The interfering RNA of (N) consists of a sequence comprising SEQ ID NO:14 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:14, and a sense strand comprising SEQ ID NO:32 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:32, and an antisense strand shown in seq id no.

The interfering RNA of (O) consists of a sequence comprising SEQ ID NO:15 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:15, and a sense strand comprising SEQ ID NO:33 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:33, and an antisense strand shown in seq id no.

The interfering RNA of (P) consists of a sequence comprising SEQ ID NO:16 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:16, and a sense strand comprising SEQ ID NO:34 or the antisense strand of the nucleotide sequence shown as SEQ ID NO: 34.

The interfering RNA of (Q) consists of a sequence comprising SEQ ID NO:17 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:17, and a sense strand comprising SEQ ID NO:35 or an antisense strand of the nucleotide sequence shown as SEQ ID NO:35, and an antisense strand shown in seq id no.

The interfering RNA of (R) consists of a sequence comprising SEQ ID NO:18 or a sense strand of a nucleotide sequence as set forth in SEQ ID NO:18, and a sense strand comprising SEQ ID NO:36 or the antisense strand of the nucleotide sequence shown as SEQ ID NO:36, and an antisense strand shown in seq id no.

Specific sense strand and antisense strand pairing information is shown in table 1.

In one embodiment of the present invention, the interfering RNA includes one or more of the above (A), (B), (C), (D), (E), (F), (H), (I), (K), (L), (M), (O), (P) or (Q) combinations.

In one embodiment of the present invention, the interfering RNA comprises one or a combination of two or more of (E), (F), (H), (I), (K), (L), (M), (O) or (P) above.

In one embodiment of the present invention, the interfering RNA comprises one or a combination of two or more of (F), (I), (K), (L), (O) or (P) above.

In one embodiment of the invention, the interfering RNA comprises one, two or three of (K), (L) or (P) above.

In one embodiment of the invention, the interfering RNA comprises one or both of (L) or (P) above.

Preferably, the interfering RNA includes, but is not limited to, one or a combination of two or more of siRNA, dsRNA, shRNA, aiRNA, miRNA.

Wherein, the siRNA is a double-stranded RNA with the length of 19-23nt, after entering cells, the siRNA is processed by a series of enzymes to form a complex with RISC (RNA-induced silencing complexes), and the target gene is specifically targeted and silenced by a base complementary pairing mode.

In one embodiment of the invention, the interfering RNA is siRNA.

The interfering RNA also contains a hanging base. For example, it contains 1 to 10 dangling bases, and more preferably 2 to 4 dangling bases.

Preferably, the end (preferably the 3' end) of the sense strand and/or the antisense strand of the interfering RNA (preferably siRNA) molecule may also have n dangling bases (Over-hang) to increase the activity of the interfering RNA. Wherein the nucleobases may be identical or different deoxynucleosides (e.g., deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU), etc.), and n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In a specific embodiment of the invention, the interference RNA sense strand and/or antisense strand terminal with 2 hanging base.

In a specific embodiment of the invention, the interfering RNA sense strand and/or antisense strand end with 2 identical or different hanging bases.

In one embodiment of the invention, the end of the sense strand and/or the antisense strand of the interfering RNA is provided with 2 identical or different dangling bases, which are deoxythymidine (dT) or deoxyuridine (dU).

In one embodiment of the invention, the dangling bases are dTdT, dTdC or dUdU.

In one embodiment of the invention, the interfering RNA molecules may further comprise at least one modified nucleotide, which modified interfering RNA has better properties, such as higher stability, lower immunostimulatory properties, etc., than the corresponding unmodified interfering RNA.

The modification includes modification in the chemical structure of the base, sugar ring and/or phosphate.

Preferably, modifications of the base include, but are not limited to, 5-pyrimidine modifications, 8-purine modifications, and/or 5-bromouracil substitutions.

Preferably, the modification of the sugar ring includes, but is not limited to, substitution of 2' -OH with H, OZ, Z, halo, SH, SZ, NH ₂、NHZ、NZ₂ or CN groups, wherein Z is an alkyl group.

The alkyl group represents a hydrocarbon chain radical which is straight or branched and does not contain an unsaturated bond, and the hydrocarbon chain radical is connected with other parts of the molecule by a single bond. Typical alkyl groups contain 1 to 20 (e.g., 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, nonyl, decyl, undecyl, 1-methylundecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, and the like.

Preferably, the phosphate backbone modification includes, but is not limited to, phosphorothioate modification.

Preferably, the modifications also include nucleotides with inosine, pigtail, xanthine, 2' -methylribose, containing non-natural phosphodiester linkages (e.g., methylphosphonate, thiophosphonate) and/or peptides.

In one embodiment of the invention, the modification may be methylation, fluorination, thiophosphorylation, pseudouracil, or the like.

The interfering RNA can be prepared and obtained by any method in the prior art. Such as chemical synthesis, etc.

In a second aspect of the invention, a delivery system is provided.

Preferably, the delivery system comprises the above interfering RNA.

Preferably, the delivery system further comprises a carrier.

Preferably, the vector may be any vector suitable for delivering the above-described interfering RNA of the present invention to a target tissue or target cell or the like, such as the vectors disclosed in the prior art (e.g., chen Zhonghua, zhu Desheng, li Jun, huang Zhanqin. "non-viral siRNA vector research progress". Chinese pharmacological bulletin. 2015, 31 (7): 910-4; wang Rui, qu Bingnan, yang. "siRNA-loaded nanoformulation research progress". Chinese pharmacy. 2017, 28 (31): 4452-4455).

In one embodiment of the invention, the vector is a viral vector, preferably, the viral vector includes but is not limited to a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxviral vector, a herpesviral vector, and the like.

In a specific embodiment of the present invention, the vector is a non-viral vector, preferably, the non-viral vector includes, but is not limited to, one or a combination of two or more of a liposome, a Lipid Nanoparticle (LNP), a polymer, a polypeptide, an antibody or an aptamer, and more preferably, the non-viral vector includes a Lipid Nanoparticle (LNP). Wherein, the weight ratio of the interfering RNA to the non-viral vector can be 1: any one of the values 1 to 50, preferably 1: any of the values 1-10 (e.g., 1:1, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50).

Preferably, the above lipid nanoparticle/liposome comprises: one or more of cationic lipid, neutral lipid, polyethylene glycol lipid, steroid lipid and anionic lipid.

Further preferably, the cationic lipid comprises: octadecyl amide (SA), lauryl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, myristyl trimethyl ammonium bromide, dimethyl Dioctadecyl Ammonium Bromide (DDAB), [ (4-hydroxybutyl) azadialkyl ] bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP), 1, 2-bis- (9Z-octadecyl) -3-trimethylammonio-propane and 1, 2-di-hexadecyl-3-trimethylammonio-propane, 3β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol (DC cholesterol), dimethyl Dioctadecyl Ammonium (DDA), 1, 2-dimyristoyl-3-trimethylammonio-propane (AP), dipalmitoyl (C16: 0) trimethylammonio-propane (DPP), diacyl trimethylammonio-propane (DSTAP), N- [1- (2, 3-propionyloxy) -3-trimethylammonio-propane and 1, 2-dioleoyl-3-dioleoyl-N, DMT-carbamoyl ] cholesterol (DC cholesterol), dimethyl dioctadecyl ammonium chloride (DDA), 1, 2-dioleoyl-Dioctadecyl Ammonium Chloride (DACs), dimethyl dioctadecyl ammonium chloride (DCAC) and dimethyl dioctadecyl ammonium chloride (DCAC), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-dioleyloxy-3-dimethylaminopropane (DLinDMA), 1, 2-ditetradecanoyl-3-dimethylammonium-propane and 1, 2-dioctadecanoyl-3-dimethylammonium-propane, 1, 2-dioleoyl-c-

(4' -Trimethylammonium) -butyryl-sn-glycerol (DOTB), dioctadecyl amide-alanyl spermine, SAINT-2, polycationic lipid 2, 3-dioleoyloxy-N- [2 (spermine-carboxamide) ethyl ] -N, N-dimethyl-1-propylammonium trifluoroacetate (DOSPA),

(SM-102)、

(JK-102-CA) or

(JK-0315-CA) or a combination of two or more thereof.

Preferably, the cationic lipid is a steroid-cationic lipid compound:

The structure of the compound is as follows:

/>

Further preferably, the neutral lipid comprises: 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE).

Further preferably, the polyethylene glycol lipid comprises: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159) 1, 2-dimyristoyl-sn-glycerylmethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxy propyl-3-amine (PEG-c-DMA),

One or a combination of two or more of the following;

Wherein n is selected from integers of 20-300, such as 20, 30, 50, 80, 100, 150, 200, 250, 300, etc.

Preferably, the polyethylene glycol lipid is a polyethylene glycol lipid of a single molecular weight, preferably, the polyethylene glycol lipid comprises:

one or a combination of two or more of them.

Further preferably, the anionic liposome comprises: one or more of dioleoyl phosphatidyl glycerol, dioleoyl phosphatidyl ethanolamine and the like.

Further preferably, the steroid lipid comprises: oat sterol, beta-sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, cholesterol, fecal sterol, dehydrocholesterol, desmosterol, dihydroergocalcitol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, algae sterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, or lithocholic acid.

In one embodiment of the invention, the lipid nanoparticle comprises the interfering RNA described above, as well as a polyethylene glycol lipid compound, a cationic lipid (excluding steroid-cationic lipid compounds), a steroid lipid or a neutral lipid.

Preferably, the molar ratio of the polyethylene glycol lipid compound, the cationic lipid, the steroid lipid and the neutral lipid in the lipid nanoparticle is (0.5-5): (30-55): (30-55): (5-20). Preferably, the molar ratio of the polyethylene glycol lipid compound, the cationic lipid, the steroid lipid and the neutral lipid is (1-5): (35-50): (40-50): (8-15).

In one embodiment of the invention, the lipid nanoparticle comprises the interfering RNA as described above, as well as a steroid-cationic lipid compound, a second lipid and a polyethylene glycol lipid. Wherein the second lipid is selected from the group consisting of: neutral lipids, zwitterionic lipids or anionic lipids.

Preferably, said steroid-cationic lipid in said lipid nanoparticle: second lipid: the polyethylene glycol lipid molar ratio is (10-30): 60-80): 10-25, preferably (20-30): 60-70): 10-20.

Preferably, the polymer may be a synthetic polymer (e.g., polyethylenimine, cyclodextrin, etc.) or a natural polymer (e.g., chitosan, telogen, etc.), or a mixture thereof.

Preferably, the polypeptide may be a Cell Penetrating Peptide (CPP) (e.g., protamine, tat peptide, transportan peptide, peneartin peptide, oligoarginine peptide, etc.).

Preferably, the antibody may be a single chain antibody (e.g., scFv-tp, scFv-9R, etc.).

The delivery system of the present application can also encapsulate various beneficial components of the human body and deliver them directly into cells for use in a faster and better manner to produce the desired effect.

In a third aspect of the invention, a cell is provided.

Preferably, the cells contain the interfering RNA or the delivery system.

Preferably, the expression level of mRNA of the gene encoding complement C3 factor in the cell and/or the expression level of complement C3 factor is inhibited.

Preferably, the cell may be any cell that expresses complement C3 factor.

Further preferably, the cells include, but are not limited to, one or more of adipocytes, myeloid cells, tumor cells, macrophages, liver cells, T cells, monocytes, and the like.

In one embodiment of the invention, the cell is a 293T cell.

In a fourth aspect of the present invention, there is provided a method for producing a cell according to the third aspect.

Preferably, the preparation method includes the interfering RNA or the delivery system into cells.

In a specific embodiment of the invention, the construction method comprises constructing a vector comprising the interfering RNA described above, and/or the delivery system according to the second aspect described above, and then introducing into the cell.

In a fifth aspect of the invention, there is provided a lipid nanoparticle comprising the interfering RNA described above.

Preferably, the lipid nanoparticle further comprises one or more of a polyethylene glycol lipid compound, a cationic lipid, a steroid lipid or a neutral lipid.

Wherein the polyethylene glycol lipid compound, the cationic lipid, the steroid lipid and the neutral lipid are defined as in the second aspect of the application.

In one embodiment of the invention, the lipid nanoparticle comprises the interfering RNA as described above, as well as polyethylene glycol lipid compounds, cationic lipids (excluding steroid-cationic lipid compounds), steroid lipids and neutral lipids.

The lipid nanoparticle of the present application can also encapsulate various beneficial components of the human body, and deliver the beneficial components directly into cells to play a role, so that the expected effect can be produced more quickly and better.

The lipid nanoparticle can be prepared by adopting a lipid nanoparticle preparation method conventional in the art, such as a high-pressure homogenization method, an emulsion precipitation method, an ultrasonic dispersion method and the like.

In a sixth aspect of the invention, there is provided a medicament or kit.

Preferably, the medicament or kit comprises the interfering RNA as described above, the delivery system as described above, the cells as described above and/or the cells obtained by the preparation method as described above.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the pharmaceutically acceptable excipients include, but are not limited to, carriers, diluents, binders, lubricants, wetting agents, and the like.

Preferably, the drug is administered by a method including, but not limited to, oral administration, enteral administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intra-ocular administration, orbital administration, retrobulbar administration, retinal administration, choroidal administration, intravitreal injection, intrathecal injection, and the like.

Preferably, the pharmaceutical dosage forms include, but are not limited to, tablets, capsules, pills, injections, inhalants, lozenges, suppositories, emulsions, microemulsions, sub-microemulsions, nanoparticles, gels, powders, suspoemulsions, creams, jellies, sprays, and the like. The various dosage forms of the medicament can be prepared according to the conventional production method in the pharmaceutical field.

Preferably, the mass content of the interfering RNA, the delivery system or the cells in the medicament may be 1-100%, e.g 1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.5％、100％.

In a seventh aspect of the invention, there is provided an antibody-nucleic acid conjugate comprising an interfering RNA as described above or a delivery system as described above or a lipid nanoparticle as described above.

The antibody-nucleic acid conjugate medicament comprises one or more interfering RNAs.

The antibody and the nucleic acid in the antibody-nucleic acid conjugate medicament can be directly connected or connected through a connecting group or a connecting peptide.

The general formula (I) of the antibody-nucleic acid coupling medicine is:

/>

Wherein R is ribonucleic acid (RNA), preferably siRNA, having the features of the first aspect of the invention;

x1 is an integer from 1 to 144; preferably, x1 is an integer from 1 to 9; more preferably, x1 is an integer from 1 to 3;

x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2;

Ab is an antibody, a protein or a polypeptide;

L is a linking unit linking Ab and R.

Preferably, the L moiety has the structure of formula (II):

Wherein,

In the formula II, x3 is selected from integers of 1-12; preferably 1 to 3;

In the formula II, x4 is selected from integers of 1-12; preferably 1 to 3;

P ₁、P₂ are the same or different polyethylene glycol residues;

L ₁ is a linking unit linking Ab and P ₁;

L ₂ is a linking unit connecting P ₂ and R;

A ₁ is a connection unit connecting between P ₁ and P ₂;

Preferably, said P ₁、P₂ is independently selected from the group consisting of linear, Y-type, multi-branched polyethylene glycol residues;

Preferably, when said P ₁、P₂ is a single molecular weight polyethylene glycol, the molecular weight is 88-4400Da, more preferably said P ₁、P₂ is 176-1056Da;

Preferably, when the P ₁、P₂ is a non-single molecular weight polyethylene glycol, the molecular weight is 1000Da-40kDa;

More preferably, the molecular weight of P ₁、P₂ is 2000Da to 10kDa.

Preferably, the L ₁ is a linking group selected from the group consisting of linear or branched C _1-12 alkylene, C _6-12 arylene, C _3-12 cycloalkylene, -S-, -O-, -S-S-、、/> One or a combination of two or more groups;

The straight or branched chain C _1-12 chain alkylene, C _6-12 arylene or C _3-12 cycloalkylene is substituted with-H, -F, -Cl, -Br, -I-O-, -S-, -SO ₂、-NO₂、C_1-12 -alkanyl, C _3-12 -cycloalkyl, C _6-12 -aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more groups of the group are substituted; /(I)

Preferably, the L ₂ is a linking group selected from the group consisting of linear or branched C _1-12 alkylene, C _6-12 arylene, C _3-12 cycloalkylene, -S-, -O-, -S-S-、/>One or a combination of two or more groups;

The straight or branched chain C _1-12 chain alkylene, C _6-12 arylene or C _3-12 cycloalkylene is substituted with-H, -F, -Cl, -Br, -I-O-, -S-, -SO ₂、-NO₂、C_1-12 -alkanyl, C _3-12 -cycloalkyl, C _6-12 -aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, A group of one or more groups;

Preferably, the L ₁ is an amide bond, a hydrazone bond, and a mercapto-maleimide bond;

more preferably, the L ₁ is an amide bond;

preferably, the L ₂ is a disulfide bond and a thiol-maleimide bond;

More preferably, the L ₂ is a disulfide bond.

Preferably, the A ₁ is a linking group linking P ₁ to P ₂ selected from the group consisting of linear or branched C _1-12 alkylene, C _6-12 arylene, C _3-12 cycloalkylene, -S-, -O-, -S-S-、One or a combination of two or more groups;

The straight or branched chain C _1-12 chain alkylene, C _6-12 arylene or C _3-12 cycloalkylene is substituted with-H, -F, -Cl, -Br, -I-O-, -S-, -SO ₂、-NO₂、C_1-12 -alkanyl, C _3-12 -cycloalkyl, C _6-12 -aralkyl, substituted or unsubstituted heterocyclyl or substituted or unsubstituted heterocyclylalkyl, One or more groups.

Further preferred, the antibody-nucleic acid conjugate drug is of formula (IV):

the L ₁ is a linking group selected from the group consisting of linear or branched C _1-12 alkylene, C _6-12 arylene, C _3-12 cycloalkylene, -S-, -O-, -S-S-、/> One or a combination of two or more groups;

Preferably, P ₁ is the same or different polyethylene glycol residue;

Preferably, when said P ₁ is a single molecular weight polyethylene glycol, the molecular weight is 88-4400Da, more preferably said P ₁ is 176-1056Da;

Preferably, when the P ₁ is a non-single molecular weight polyethylene glycol, the molecular weight is 1000Da-40kDa; more preferably, the molecular weight of P ₁ is 2000Da to 10kDa.

The L ₂ is a linking group selected from the group consisting of linear or branched C _1-12 alkylene, C _6-12 arylene, C _3-12 cycloalkylene, -S-, -O-, -S-S-、One or a combination of two or more groups;

x2 is an integer from 1 to 8; preferably, x2 is an integer from 1 to 2.

In one embodiment of the invention, the antibody-nucleic acid conjugate drug has the following structure:

Said n ₁ is selected from integers from 4 to 100, preferably from 4 to 24; n1 may be a constant value or an average value.

The R is ribonucleic acid (RNA), preferably siRNA, having the features described in the first aspect of the invention.

the n ₁、n₂ is independently selected from integers from 4 to 100, preferably from 4 to 24. n1 and n2 may be constant values or average values.

Preferably, the Ab is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, and antibody fusion fragments.

The antibody may be a single domain antibody or a single chain antibody.

Further preferred, ab is a monoclonal antibody; more preferably, the monoclonal antibody is reactive with an antigen or epitope thereof associated with cancer, malignant cells, infectious organisms, or autoimmune diseases.

In a specific embodiment of the invention, the Ab is selected from: an anti-HER 2 antibody, an anti-EGFR antibody, an anti-PMSA antibody, an anti-VEGFR antibody, an anti-CD 30 antibody, an anti-CD 22 antibody, an anti-CD 56 antibody, an anti-CD 29 antibody, an anti-GPNMB antibody, an anti-CD 138 antibody, an anti-CD 74 antibody, an anti-ENPP 3 antibody, an anti-Nectin-4 antibody, an anti-EGFR viii antibody, an anti-SLC 44A4 antibody, an anti-mesothelin antibody, an anti-ET 8R antibody, an anti-CD 37 antibody, an anti-CEACAM 5 antibody, an anti-CD 70 antibody, an anti-MUC 16 antibody, an anti-CD 79b antibody, an anti-MUC 16 antibody, an anti-MUC 1 antibody, an anti-CD 3 antibody, an anti-CD 28 antibody, an anti-CD 38 antibody, an anti-CD 19 antibody, an anti-PD-L1 antibody, or an anti-4-1 BB antibody, or the like.

In an eighth aspect of the present invention, there is provided a method of inhibiting complement factor C3.

Preferably, the inhibition method comprises the use of the above interfering RNA, the above delivery system, the above cells, the above preparation method obtained cells, the above drugs or kits, and/or the above antibody-nucleic acid conjugate drug.

In a ninth aspect of the invention, there is provided the use of an interfering RNA as described above, a delivery system as described above, a cell obtained by the above construction method, a drug or kit as described above, an antibody-nucleic acid conjugate drug as described above, and/or an inhibition method as described above.

Preferably, the application comprises:

a) Use in the manufacture of a product for the prevention and/or treatment of an ocular disease;

b) The application in preparing products for preventing and/or treating tumor;

c) Use in the preparation of a product for the prevention and/or treatment of inflammation;

D) The application in preparing products for preventing and/or treating ischemic cerebrovascular diseases;

E) Use in the manufacture of a product for the prevention and/or treatment of kidney disease;

f) Use in inhibiting complement C3 factor expression-related diseases;

g) Use in the preparation of a medicament for preventing and/or treating diseases associated with expression of complement C3 factor.

Preferably, the ocular diseases include, but are not limited to, macular degeneration diseases such as all stages of age-related macular degeneration (AMD), diabetic retinopathy and other ischemia-related retinopathies, uveitis, diabetic Retinopathy (DR), endophthalmitis, diabetic macular edema, pathologic myopia, von Hippel-Lindau disease, histoplasmosis of the eye, central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization. Among them, age-related macular degeneration (AMD) includes non-exudative/non-neovascular/dry AMD and exudative/wet/neovascular AMD, wherein the former includes metaphase (intermediate) dry AMD or geographic atrophy (geographic atrophy, GA) and the like; exudative AMD, also known as neovascular or wet AMD, is characterized by Choroidal Neovascularization (CNV), and additionally non-exudative AMD can include drusen, molluscum, geographic atrophy, and/or pigment clumping, among others.

Preferably, the tumors include, but are not limited to, tumors of the digestive tract (oral cancer, tongue cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), tumors of the nervous system [ glioma (e.g., glioma), neuroepithelial tumor, schwannoma, astrocytoma, neurofibroma (e.g., neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastasis, etc. ], tumors of the respiratory tract (nasopharyngeal carcinoma, laryngeal cancer, bronchogenic carcinoma, lung cancer, etc.), tumors of the urinary system (e.g., prostate cancer, renal cell carcinoma, bladder cancer, etc.), tumors of the reproductive system (breast cancer, cervical cancer, ovarian cancer, choriocarcinoma, etc.), tumors of the blood lymphatic system [ multiple myeloma, mesothelioma, myelodysplastic syndrome, lymphomas (e.g., non-hodgkin lymphoma, hodgkin's lymphoma, cutaneous T cell lymphoma), leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, etc.), tumors of the thymus, etc. ], tumors of the skin system (e.g., skin cancer, epidermoid carcinoma, melanoma, etc.), and the like), sarcomas of the head and neck, etc.

Preferably, the inflammation includes, but is not limited to, acute inflammation, and also includes chronic inflammation. In particular, including but not limited to degenerative, exudative, proliferative, etc., including but not limited to severe burns, endotoxemia, septic shock, paroxysmal sleep hemoglobinuria, adult respiratory distress syndrome, myasthenia gravis, sjogren's syndrome, lupus erythematosus, hemodialysis, anaphylactic shock, severe asthma, angioedema, crohn's disease, sickle cell anemia, post streptococcal glomerulonephritis, pancreatitis, enteritis, vasculitis, adverse drug reactions, drug allergies, IL-2 induced vascular leakage syndrome, or radiographic (contrast) contrast agent allergies, etc.

Preferably, the kidney disease includes, but is not limited to IgA nephropathy, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, C3 glomerulopathy, renal atrophy, lupus nephritis and/or paroxysmal sleep hemoglobinuria, and the like.

In a tenth aspect of the invention, there is provided a method of reducing expression of a gene encoding complement C3 factor in an individual.

Preferably, the method comprises the step of administering to the individual a therapeutically effective amount of the interfering RNA, the delivery system, the cell, the lipid nanoparticle, the drug or the antibody-nucleic acid conjugate drug.

In an eleventh aspect of the invention, there is provided a method of inhibiting complement factor C3 encoding gene and/or detecting complement factor C3 expression.

Preferably, the method comprises the step of administering to the individual a therapeutically effective amount of the interfering RNA, the cell, the delivery system, the lipid nanoparticle, the drug or the antibody-nucleic acid conjugate drug.

In a twelfth aspect of the present invention, there is provided a method for preventing and/or treating a disease associated with complement C3 factor, comprising the step of administering to an individual a therapeutically effective amount of the interfering RNA, the cell, the delivery system, the lipid nanoparticle, the drug or the antibody-nucleic acid conjugate drug.

Preferably, the complement C3 factor-associated disease includes, but is not limited to, ocular disease, tumor, ischemic cerebrovascular disease, kidney disease, and the like. The eye diseases, tumor, ischemic cerebrovascular diseases and kidney diseases are the same as defined in the seventh aspect.

The "interfering RNAs" of the present invention, including single-stranded RNAs (e.g., mature mirnas, ssRNAi oligonucleotides, ssDNAi oligonucleotides) or double-stranded RNAs (e.g., siRNA, dsRNA, shRNA, aiRNA, or precursor mirnas), are capable of reducing or inhibiting expression of a target gene or sequence (e.g., by mediating degradation and/or inhibiting translation of mRNA complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence.

Wherein the siRNA is a small interfering RNA, each strand of the molecule of which comprises nucleotides of about 15 to about 60 in length (e.g., nucleotides of about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 in length, or nucleotides of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 in length). In a specific embodiment, the siRNA can be chemically synthesized. The siRNA molecules of the invention are capable of silencing expression of a target sequence in vitro and/or in vivo. In some embodiments, the siRNA may not contain modified nucleotides; in other embodiments, the siRNA comprises at least one modified nucleotide, e.g., the siRNA comprises one, two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides in the double-stranded region. dsRNA or precursor RNA molecules include any precursor molecule that is processed in vivo by an endonuclease to produce an active siRNA. shRNA is a small hairpin RNA or short hairpin RNA, comprising a short RNA sequence that produces tight hairpin turns (hairpin turns) that can be used to silence gene expression by RNA interference, and shRNA hairpin structures can be cleaved by cellular machinery into sirnas. mirnas (micrornas) are single stranded RNA molecules of about 21-23 nucleotides in length that regulate gene expression.

The invention of the "inhibition of target genes or sequences of expression" refers to the invention of interfering RNA (e.g., siRNA) silencing, reducing or inhibiting target genes (e.g., complement C3 factor encoding genes) expression ability.

The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

The term "therapeutically effective amount" as used herein refers to an amount or dose of a product of the invention that provides a desired effect (e.g., treating, preventing a disease or inhibiting expression of complement C3 factor) after administration to a subject or cells or organs thereof in single or multiple doses.

The term "lipid" as used herein refers to a group of organic compounds including, but not limited to, esters of fatty acids, and is generally characterized as poorly soluble in water but soluble in many organic solvents.

The term "cationic lipid" as used herein refers to a lipid molecule capable of being positively charged.

The term "neutral lipid" as used herein refers to lipid molecules that are uncharged, non-phosphoglycerides.

The term "polyethylene glycol lipid" as used herein refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.

The term "lipid nanoparticle" as used herein refers to particles having at least one nanoscale size, which comprises at least one lipid.

The term "delivery system" as used herein refers to a formulation or composition that modulates the spatial, temporal and dose distribution of a biologically active ingredient within an organism.

The term "comprising" or "comprises" as used herein is an open-ended writing that includes the specified components or steps described, as well as other specified components or steps that are not materially affected. When used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the same or similar activity as the original sequence.

All combinations of items to which the term "and/or" is attached "in this description shall be taken to mean that the respective combinations have been individually listed herein. For example, "a and/or B" includes "a", "a and B", and "B". Also for example, "A, B and/or C" include "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".

An "individual" as described herein may be a human or non-human animal, such as a non-human mammal. The "non-human mammal" may be a wild animal, zoo animal, economic animal, pet animal, laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, a pig, cow, sheep, horse, donkey, fox, raccoon dog, marten, camel, dog, cat, rabbit, mouse (e.g., rat, mouse, guinea pig, hamster, gerbil, dragon cat, squirrel) or monkey, and the like.

Drawings

Fig. 1: mass spectrum of complement C3 factor siRNA (siC 3-215);

Fig. 2: mass spectrum of complement C3 factor siRNA (siC-489);

fig. 3: mass spectrum of complement C3 factor siRNA (siC-498);

Fig. 4: complement C3 factor; mass spectrum of siRNA (siC-924);

fig. 5: mass spectrum of complement C3 factor siRNA (siC 3-1244);

fig. 6: mass spectrum of complement C3 factor siRNA (siC-1802);

fig. 7: mass spectrum of complement C3 factor siRNA (siC 3-2241);

Fig. 8: mass spectrum of complement C3 factor siRNA (siC 3-2244);

fig. 9: mass spectrum of complement C3 factor siRNA (siC 3-2269);

Fig. 10: mass spectrum of complement C3 factor siRNA (siC-2739);

Fig. 11: mass spectrum of complement C3 factor siRNA (siC 3-2859);

fig. 12: mass spectrum of complement C3 factor siRNA (siC-3165);

Fig. 13: mass spectrum of complement C3 factor siRNA (siC-3195);

Fig. 14: mass spectrum of complement C3 factor siRNA (siC-4395);

fig. 15: mass spectrum of complement C3 factor siRNA (siC-4420);

Fig. 16: mass spectrum of complement C3 factor siRNA (siC-4715);

fig. 17: mass spectrum of complement C3 factor siRNA (siC-4740);

Fig. 18: mass spectrum of complement C3 factor siRNA (siC-4752);

Fig. 19: complement C3 factor siRNA (negative control siNC) mass spectrum;

fig. 20: the relative expression levels of complement C3 factor encoding genes in the cells after use of each set of complement C3 factor sirnas;

Fig. 21: cell viability status after use of complement C3 factor siRNA for each group.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Reagents or instrumentation used in the examples:

293T cells were purchased from national biomedical experimental cell resource libraries;

Lipofectamine RNAi MAX ^TM transfection reagent was purchased from the english-switzerland (Shanghai) trade company, cat No. 13778075;

RNA extraction kit: purchased from Promega corporation under the designation LS1040:

Reverse transcription kit: purchased from Promega corporation under the accession number a2791;

TB green Premix Ex Taq II (TLI RNASEH Plus): purchased from Takara corporation under the product number RR820A.

SiC3 in the examples represents siRNA targeting complement factor C3; siNC represents a negative control.

Embodiment one: siRNA design and Synthesis

For C3 sequence (GenBank: NM_ 000064.4), 18 pairs of siRNA sequences are designed, the sequence information is shown in table 1, the mass spectrum detection result of each siRNA is shown in figures 1-18, and when in actual use, 2 hanging bases dTdT are added at the 3' end of the sense strand and/or the antisense strand of the siRNA.

Table 1: designed C3 siRNA sequence

/>

The above sequence is synthesized by Beijing qingke biotechnology limited company, and dTdT is automatically added at the end of the synthesis; a negative control sequence (siNC) was also synthesized, the sense strand UUCUCCGAACGUGUCACGUdTdT (SEQ ID NO: 37) and the antisense strand ACGUGACACGUUCGGAGAAdTdT (SEQ ID NO: 38), and the mass spectrum of siNC was shown in FIG. 19.

Embodiment two: inhibition effect of interfering RNA on complement C3 factor encoding gene

1. Cell culture and transfection

293T cells (human kidney epithelial cell line transfected with adenovirus E1A gene) were used to verify the inhibition effect of siRNA.

(1) 293T cells were cultured in DMEM medium (100U/ML PENICILLIN, 100. Mu.g/mL Streptomycin) containing 10% FBS at 37℃in a 5% CO ₂ saturated humidity incubator. Cells were seeded at 1.5×10 ⁵ cells per well in 24 well plates for overnight incubation 24h prior to the experiment.

(2) Diluting 2.5 mu L of OPTI-MEM culture medium with 2.5 mu L of each group siC or siNC (the concentration of mother solution is 10 mu M), diluting 1.5 mu L Lipofectamine RNAiMax ^TM of transfection reagent with 23.5 mu L of OPTI-MEM culture medium, standing for 5min, mixing, and gently shaking for later use. Furthermore, a Blank group is set.

(3) The cells in each well of the cell plate were changed, and a medium without antibiotic was added thereto in an amount of 450. Mu.L per well, and then 50. Mu.L of the above-mentioned mixed solution was added to each well, and finally the total volume per well was 500. Mu.L. The final concentration of siRNA (or siRNA NC) transfection was 50nM.

(4) After 48h of transfection, the 24-well plates were removed from the incubator at 37℃with 5% CO ₂ and the cells were collected for extraction of RNA for subsequent detection.

2. Extraction of RNA

(1) RNA extraction was performed using Promega RNA extraction kit. Briefly, after washing the cells with PBS, 300. Mu.L of lysis solution was added, the cells were blown up with a pipette, after sufficient lysis, 300. Mu.L of dilution was added, and then water bath was performed at 70℃for 3min. Centrifuge at 14000rpm for 10min, transfer the supernatant to a new 1.5mL Ep tube, add 300 μl absolute ethanol, mix well and add to the column. Centrifuge at 14000rpm for 1min, discard filtrate, add 600 μl wash and centrifuge again for 1min. Discarding the filtrate, adding 50 mu L of DNase I reaction solution into each hole, and standing at room temperature for 15min; adding 600 mu L of washing liquid, and centrifuging for 1min; removing filtrate, adding 600 μl of washing solution, and centrifuging for 1min again; the filtrate was discarded, centrifuged for 2min, 50. Mu.L of nuclease-free water was added to each well, and the mixture was allowed to stand at room temperature for 5min, followed by centrifugation to collect the eluted RNA.

(2) RNA quality inspection, RNA content and purity detection, and 1% agarose gel electrophoresis detection of RNA integrity.

3. Q-PCR detection flow

(1) RNA reverse transcription

The total RNA extracted from the sample was used as a template, and a reaction system shown in Table 2 was established using the Promega reverse transcription kit:

table 2: RNA reverse transcription system

Reagent(s)	Usage amount
		5×RT Buffer(oligodT)	4μL
RTase Mix	2μL
		RNA template	1μg
RNase-free H₂O	Is added to 20 mu L
		Totals to	20μL

Mixing the above systems, centrifuging to collect liquid to the bottom of the tube, reacting at 42 deg.C for 60min, and reacting at 72 deg.C for 10min; the product is the cDNA template.

(2) Fluorescent quantitative PCR detection

Using TB green Premix Ex Taq II (TLI RNASEH Plus) (Takara) reagent, a reaction system was established as shown in Table 3:

table 3: fluorescent quantitative PCR reaction system

Reagent(s)	Addition amount of
		2×SYBR Green Mix	10μL
Forward primer (10. Mu.M)	0.4μL
		Reverse primer (10. Mu.M)	0.4μL
cDNA	2μL
		RNase-free H₂O	7.2μL
Totals to	20μL

Table 4: primer sequences

PCR amplification was performed as follows, and the primer sequences are shown in Table 4.

Pre-denatured at 95 ℃ for 10min, and then enters the following circulation

*95℃10s

60℃20s

Reading board

Return is performed for a total of 40 cycles.

Preparing a melting curve: plates were read between 65℃and 95℃and stopped for 5s every 0.5 ℃.

4. Inhibition effect

Using GAPDH as an internal standard gene, and calculating the relative expression quantity of C3 mRNA by a delta Ct method; each group of mRNA expression levels were normalized to 100% of the blank group expression levels.

The relative expression levels of the mRNA of each cell group are shown in Table 5 and FIG. 20.

Table 5: c3 Relative mRNA expression levels

Note that: siNC in the table is a negative control.

The results show that the expression level of the C3 mRNA in the cells treated by the siRNA disclosed by the invention, such as siC-1244, siC-1802, siC3-2244, siC-2269, siC-2859, siC3-3165, siC3-3195, siC3-4420 and siC3-4715, is obviously reduced relative to the relative expression level of the blanc group and siNC group at 48 hours after transfection, and the screened C3 siRNA has obvious and continuous effect of inhibiting the expression of the C3 gene. Wherein siC-4420 has the best inhibiting effect.

Embodiment III: interfering RNA cytotoxicity assay

293T cells were plated in 96-well plates at a cell density of 5000/well and incubated at 37℃for 24h. Then 180 mu L of serum-free culture medium is added into each well, transfection reagents are prepared according to the specification of lipofectamine RNAiMAX according to the proportion, 20 mu L of the corresponding group of transfection reagents are respectively added into the cell holes, and the final concentration of siRNA in the cell holes is 50nM. 48h after transfection, 10. Mu.L of 5mg/mL MTT solution was added to each cell well and incubation was continued at 37℃for 4h. The medium was then discarded, 200. Mu.L of DMSO was added to each well, and absorbance was measured after shaking for 10min (A ₄₉₀). Cell viability was calculated for each group of cells using the blank as a standard.

The cytotoxicity of siRNA was examined by MTT method for siC sequences showing remarkable inhibitory effect.

Cell viability (Viability%) =100×a _Experiment/A_Control

The results are shown in Table 6 and FIG. 21.

Table 6: c3 Cell viability of groups after siRNA treatment

The results showed that the cell viability of each of siC, 3-2859, siC, 3-3165, siC, 3-4715 treated cells was 70% or more, comparable to siNC, and no cytotoxicity was considered. siC3-1244, siC3-1802, siC3-2244, siC3-2269, siC3-3195, siC3-4420 are slightly cytotoxic.

Embodiment four: LNP preparation, biological Activity and cytotoxicity evaluation

1. Preparation of LNP-siC3

Weighing SM-102.02 mg, adding 10mL of EtOH, and dissolving to obtain SM-102 mother liquor;

Weighing 249.5mg of M-DMG-2000, and dissolving 10mL of absolute EtOH to obtain PEG component mother liquor;

Weighing 79.02mg of DSPC, 10mL of anhydrous EtOH, and dissolving to obtain DSPC mother liquor;

38.66mg of cholesterol was weighed out, 10mL of absolute EtOH was used, and the resulting cholesterol mother liquor was dissolved.

Taking 64.8 mu L, DSPC mu L of SM-102 mother liquor 2160 mu L, PEG component mother liquor and 1664 mu L of cholesterol mother liquor respectively, and uniformly mixing to obtain LNP/EtOH solution;

150nmol of siRNA in Table 1 of example 1 was added to 50mM citrate buffer (pH=4.0)

6.5ML, obtaining siRNA/buffer solution;

Microfluidic preparation: LNP/EtOH solution was used in passageway 1, solution volume 2mL, flow rate 5mL/min; the siRNA/buffer solution uses a passage 2, the volume of the solution is 6mL, and the flow rate is 15mL/min;

4mL of the microfluidic preparation solution was dialyzed with PBS (pH=7.4) for 16h to obtain siRNA-LNP with a final concentration of 0.2mg/mL.

2. The effective particle diameter and zeta potential of LNP-siC3 are detected by a nanometer particle diameter instrument

The particle size and zeta potential of LNP-siC3 were measured using a NanoBrook (Bruce Haille) nanosized particle meter. Prepared LNP-siC. Mu.L was diluted to 2mL with DEPC water and checked on-line. Each sample was tested three times and particle size was expressed as the mean of the effective particle size (D50).

3. Evaluation of LNP-siC3 biological Activity

(1) 293T cell culture and plating steps are the same as in examples two, 1 and (1).

(2) The prepared LNP was diluted to 500nM with opti-MEM and gently shaken for use. Meanwhile, blank, lipofectamine RNAiMAX, NC, and lipofectamine RNAiMAX transfection groups were set (preparation method as in examples two, 1, and (2)).

(5) RNA extraction and qPCR detection were performed in the same manner as in examples two and 2-3.

The results show that LNP delivers siC sequences (siC 3-3165, siC 34715), which can achieve the same or even better results as lipofectamine RNAiMAX, C3 mRNA expression is significantly inhibited.

4. LNP-siC cytotoxicity evaluation

The procedure is as in example 3, using 293T cells.

LNP-siC3 has similar or better delivery effect than lipofectamine RNAiMAX, and the expression level of C3 mRNA in cells is obviously reduced.

Fifth embodiment: siRNA sequence modification and biological effect evaluation

1. SiC3 sequence modification

Each sequence siC is modified, methylation, fluoro, thiophosphorylation, pseudouracil and other modifications are added, and the modified sequence is synthesized by Shanghai Biotechnology Co.

2. Evaluation of biological Effect and cytotoxicity of siC modified

The biological activity of the modified sequence was detected by qPCR. Cell culture, transfection, RNA extraction, reverse transcription, qPCR were performed as in examples two and three.

The modified siC sequence has similar or better effect of inhibiting the expression of C3 mRNA than the unmodified siC sequence; meanwhile, the cytotoxicity test results show that the sequence modification does not obviously increase the cytotoxicity.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims

1. An interfering RNA targeting the complement factor C3 encoding gene, said interfering RNA comprising the sequence of SEQ ID NO:1-36 or two or more of the nucleotide sequences shown in seq id no.

2. The interfering RNA of claim 1 wherein the interfering RNA comprises a sense strand and/or an antisense strand;

The sense strand comprises SEQ ID NO:1-18 or two or more of the nucleotide sequences shown in seq id no;

The antisense strand comprises SEQ ID NO:19-36 or two or more of the nucleotide sequences shown in seq id no.

3. The interfering RNA of claim 1 or 2, wherein the interfering RNA comprises any one of the following group:

A) The sense strand comprises SEQ ID NO:1, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:19, a nucleotide sequence shown in seq id no;

b) The sense strand comprises SEQ ID NO:2, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:20, a nucleotide sequence shown in seq id no;

c) The sense strand comprises SEQ ID NO:3, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:21, a nucleotide sequence shown in seq id no;

d) The sense strand comprises SEQ ID NO:4, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:22, a nucleotide sequence shown in seq id no;

E) The sense strand comprises SEQ ID NO:5, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:23, a nucleotide sequence shown in seq id no;

f) The sense strand comprises SEQ ID NO:6, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:24, a nucleotide sequence shown in seq id no;

G) The sense strand comprises SEQ ID NO:7, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:25, a nucleotide sequence shown in seq id no;

H) The sense strand comprises SEQ ID NO:8, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:26, a nucleotide sequence shown in seq id no;

i) The sense strand comprises SEQ ID NO:9, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:27, a nucleotide sequence set forth in seq id no;

j) The sense strand comprises SEQ ID NO:10, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:28, a nucleotide sequence shown in seq id no;

k) The sense strand comprises SEQ ID NO:11, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:29, a nucleotide sequence set forth in seq id no;

L) sense strand comprises SEQ ID NO:12, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:30, a nucleotide sequence shown in seq id no;

M) sense strand comprises SEQ ID NO:13, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:31, a nucleotide sequence shown in seq id no;

n) sense strand comprises SEQ ID NO:14, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:32, a nucleotide sequence shown in seq id no;

O) sense strand comprises SEQ ID NO:15, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:33, a nucleotide sequence set forth in seq id no;

P) sense strand comprises SEQ ID NO:16, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:34, a nucleotide sequence shown in seq id no;

q) the sense strand comprises SEQ ID NO:17, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:35, a nucleotide sequence shown in seq id no;

r) sense strand comprises SEQ ID NO:18, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no.

4. The interfering RNA of any one of claims 1-3 wherein the interfering RNA further comprises a dangling base;

preferably, the interfering RNA comprises 1-10 dangling bases, more preferably 2-4 dangling bases;

preferably, the overhang base is located at the 3' end of the sense strand and/or antisense strand of the interfering RNA;

preferably, the hanging base is a deoxynucleoside;

More preferably, the hanging base is dTdT, dTdC or dUdU.

5. The interfering RNA of any one of claims 1-4 wherein the interfering RNA comprises one or a combination of more than two of siRNA, dsRNA, shRNA, aiRNA or miRNA;

preferably, the interfering RNA is siRNA.

6. The interfering RNA of any one of claims 1-5 wherein the interfering RNA further comprises at least one modification; the modification includes modification on a base, sugar ring and/or phosphate backbone;

preferably, modifications of the base include, but are not limited to, pyrimidine modifications at position 5, purine modifications at position 8, pseudouracil modifications, and/or 5-bromouracil substitutions;

Preferably, the modification of the sugar ring includes, but is not limited to, substitution of 2' -OH with H, OZ, Z, halo, SH, SZ, NH ₂、NHZ、NZ₂ or CN groups, wherein Z is an alkyl group;

7. The interfering RNA of claim 6 wherein the modification further comprises a nucleotide having inosine, pigtail, xanthine, 2' -methylribose, a non-natural phosphodiester linkage (e.g., methylphosphonate, phosphorothioate) and/or a peptide.

8. A delivery system for an interfering RNA according to any one of claims 1 to 7, wherein the delivery system comprises the interfering RNA according to any one of claims 1 to 7 and a vector.

9. The delivery system of claim 8, wherein the vector is a viral vector or a non-viral vector;

Preferably, the viral vector comprises: one or a combination of two or more of lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, poxviral vectors or herpesviral vectors;

Preferably, the non-viral vector comprises: any one or a combination of two or more of a liposome, a lipid nanoparticle, a polymer, a polypeptide, an antibody, an aptamer or N-acetylgalactosamine (GalNAc).

10. The delivery system of claim 9, wherein the lipid nanoparticle/liposome comprises: one or more of cationic lipid, neutral lipid, polyethylene glycol lipid, steroid lipid or anionic lipid.

11. The delivery system of claim 10, wherein the cationic lipid comprises: octadecyl amide (SA), lauryl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, myristyl trimethyl ammonium bromide, dimethyl Dioctadecyl Ammonium Bromide (DDAB), [ (4-hydroxybutyl) azadialkyl ] bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), 1, 2-dioleoyloxy-3- (trimethylammonio) propane (DOTAP), 1, 2-bis- (9Z-octadecyl) -3-trimethylammonio-propane and 1, 2-di-hexadecyl-3-trimethylammonio-propane, 3β - [ N- (N ', N' -dimethylaminoethane) -carbamoyl ] cholesterol (DC cholesterol), dimethyl Dioctadecyl Ammonium (DDA), 1, 2-dimyristoyl-3-trimethylammonio-propane (AP), dipalmitoyl (C16: 0) trimethylammonio-propane (DPP), diacyl trimethylammonio-propane (DSTAP), N- [1- (2, 3-propionyloxy) -3-trimethylammonio-propane and 1, 2-dioleoyl-3-dioleoyl-N, DMT-carbamoyl ] cholesterol (DC cholesterol), dimethyl dioctadecyl ammonium chloride (DDA), 1, 2-dioleoyl-Dioctadecyl Ammonium Chloride (DACs), dimethyl dioctadecyl ammonium chloride (DCAC) and dimethyl dioctadecyl ammonium chloride (DCAC), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-dioleyloxy-3-dimethylaminopropane (DLinDMA), 1, 2-ditetradecanoyl-3-dimethylammonium-propane and 1, 2-dioctadecanoyl-3-dimethylammonium-propane, 1, 2-dioleoyl-c- (4' -trimethylammonium) -butyryl-sn-glycerol (DOTB), dioctadecanoyl-alanyl-spermine, SAINT-2, polycationic lipids 2, 3-dioleoyloxy-N- [2 (spermine-carboxamide) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA),

One or a combination of two or more of them.

12. The delivery system of claim 10, wherein the neutral lipid comprises: 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho- (1' -rac-glycerol) (DOPG), oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), or distearoyl phosphatidylethanolamine (DSPE).

13. The delivery system of claim 10, wherein the polyethylene glycol lipid comprises: 2- [ (polyethylene glycol) -2000] -N, N-tetracosylacetamide (ALC-0159) 1, 2-dimyristoyl-sn-glycerylmethoxy polyethylene glycol (PEG-DMG), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ] (PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE) or PEG-1, 2-dimyristoyloxy propyl-3-amine (PEG-c-DMA),

One or a combination of two or more of them;

Wherein n is an integer from 20 to 300.

14. The delivery system of claim 10, wherein the polyethylene glycol lipid is a single molecular weight polyethylene glycol lipid, preferably the polyethylene glycol lipid comprises:

one or a combination of two or more of them.

15. The delivery system of claim 10, wherein the cationic lipid is a steroid-cationic lipid compound:

The structure of the compound is as follows:

16. the delivery system of claim 10, wherein the anionic liposome comprises: one or more of dioleoyl phosphatidyl glycerol and dioleoyl phosphatidyl ethanolamine.

17. The delivery system of claim 10, wherein the steroid lipid comprises: oat sterol, beta-sitosterol, campesterol, ergocalcitol, campesterol, cholestanol, cholesterol, fecal sterol, dehydrocholesterol, desmosterol, dihydroergocalcitol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, algae sterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, or lithocholic acid.

18. A cell comprising the interfering RNA of any one of claims 1-7 or the delivery system of any one of claims 8-17.

19. A method of preparing a cell, comprising introducing into the cell an interfering RNA of any one of claims 1-7 or a delivery system of any one of claims 8-17.

20. A medicament or kit comprising one or a combination of two or more of the interfering RNA of any one of claims 1-7, the delivery system of any one of claims 8-17, the cell of claim 18 or the cell obtained by the method of preparation of claim 19;

preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials;

Further preferably, the pharmaceutically acceptable auxiliary materials comprise one or more than two of pharmaceutical carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and fragrances.

21. An antibody-nucleic acid conjugate comprising the interfering RNA of any one of claims 1-7 or the delivery system of any one of claims 8-17.

22. A method of inhibiting complement factor C3, comprising using one or more of the interfering RNA of any one of claims 1-7, the delivery system of any one of claims 8-17, the cell of claim 18, the cell obtained by the method of preparation of claim 19, the drug or kit of claim 20, or the antibody-nucleic acid conjugate drug of claim 21.

23. Use of the interfering RNA of any one of claims 1-7, the delivery system of any one of claims 8-17, the cell of claim 18, the cell obtained by the method of preparation of claim 19, the drug or kit of claim 20, the antibody-nucleic acid conjugate drug of claim 21, or the inhibition method of claim 22, wherein the use comprises:

b) The application in preparing products for preventing and/or treating tumor;

F) Use in the preparation of a medicament for preventing and/or treating diseases associated with expression of complement C3 factor.

24. The use according to claim 23, wherein the ocular diseases include macular degeneration diseases such as all stages of age-related macular degeneration (AMD), diabetic retinopathy and other ischemia-related retinopathies, uveitis, diabetic Retinopathy (DR), endophthalmitis, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, central Retinal Vein Occlusion (CRVO), corneal neovascularization and retinal neovascularization.

25. The use according to claim 23, wherein said tumor comprises a tumor of the digestive tract (oral cancer, tongue cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, etc.), a tumor of the nervous system [ glioma (e.g. glioma), neuroepithelial tumor, schwannoma, astrocytoma, neurofibroma (e.g. neurofibrosarcoma), ependymoma, medulloblastoma, meningioma, brain metastasis, etc. ], a tumor of the respiratory tract (nasopharyngeal cancer, laryngeal cancer, bronchial cancer, lung cancer, etc.), a tumor of the urinary system (e.g. prostate cancer, renal cell cancer, bladder cancer, etc.), a tumor of the reproductive system (breast cancer, cervical cancer, ovarian cancer, placental villous cancer, etc.), a tumor of the blood lymphatic system [ multiple myeloma, mesothelioma, myelodysplastic syndrome, lymphoma (e.g. non-hodgkin's lymphoma, cutaneous T cell lymphoma), leukemia (e.g. chronic myelogenous leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, etc.), thymus cancer, etc. ] tumor of the skin system (e.g. skin cancer, epidermoid carcinoma, melanoma, etc.), head and neck cancer, etc.

26. The use according to claim 23, wherein the inflammation comprises acute inflammation and/or chronic inflammation;

Preferably, the inflammation comprises degenerative inflammation, exudative inflammation, proliferative inflammation, severe burns, endotoxemia, infectious shock, paroxysmal sleep hemoglobinuria, adult respiratory distress syndrome, myasthenia gravis, sjogren's syndrome, lupus erythematosus, hemodialysis, anaphylactic shock, severe asthma, angioedema, crohn's disease, sickle cell anemia, post-streptococcal glomerulonephritis, pancreatitis, enteritis, vasculitis, adverse drug reactions, drug allergies, IL-2 induced vascular leakage syndrome, or radiographic (contrast) contrast agent allergies.

27. The use according to claim 23, wherein said kidney disease comprises IgA nephropathy, atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, C3 glomerulopathy, renal atrophy, lupus nephritis and/or paroxysmal sleep haemoglobinuria.