WO2025113592A1 - Rna inhibitor for inhibiting gene expression of complement system and use thereof - Google Patents

Abstract

An RNA inhibitor for inhibiting gene expression of a complement system, or a pharmaceutically acceptable salt thereof. The structure of the RNA inhibitor further contains vector structures 5'MVIP and 3'MVIP. The provided RNA inhibitor interferes with the translation template function of the mRNA of the complement system, and continuously and efficiently inhibits the gene expression of the complement system, so that CFB, C5 and C3 protein levels of the complement system in blood are continuously reduced. The RNA inhibitor can be used for treating and/or preventing diseases associated with an increased level of the complement system.

Description

RNA inhibitor for inhibiting complement system gene expression and its application Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to an RNA inhibitor for inhibiting complement system gene expression and application thereof.

Background Art

RNAi

RNAi (RNA interference) was discovered in 1998 by Andrew Z. Fire and others during an antisense RNA inhibition experiment in Caenorhabditis elegans, and the process was called RNA interference. This discovery was named one of the top ten scientific advances in 2001 by Science magazine, and ranked first among the top ten scientific advances in 2002. Since then, siRNA, which uses RNA interference as a mechanism of action, has received widespread attention as a potential gene therapy drug. In 2006, Andrew Z. Fire and Craig C. Mello won the Nobel Prize in Physiology or Medicine for their contributions to the study of RNA interference mechanisms. RNAi can be triggered by double-stranded RNA (dsRNA) in many organisms, including animals, plants, and fungi. In the RNA inhibitor process, a nuclease called "Dicer" cuts or "dices" the long-chain dsRNA into small fragments of 21 to 25 nucleotides. These small fragments, called small interfering RNA (siRNA), have their antisense strands loaded onto the Argonaute protein (AGO2). AGO2 loading occurs in the RISC-loading complex, a ternary complex consisting of the Argonaute protein, Dicer, and the dsRNA binding protein (TRBP for short). During the loading process, the sense strand (Passenger strand) is cleaved and expelled by AGO2. AGO2 then uses the antisense strand to bind to mRNAs containing completely complementary sequences and then catalyzes the cleavage of these mRNAs, causing the mRNA split to lose its role as a translation template, thereby preventing the synthesis of related proteins. After cleavage, the cleaved mRNA is released, and the RISC-loading complex loaded with the antisense strand is recycled for another round of cleavage.

According to statistics, among the disease-related proteins in the human body, more than 80% of the proteins cannot be targeted by the current conventional small molecule drugs and biological macromolecule preparations, and are non-drugable proteins. Gene therapy, which aims to treat diseases through gene expression, silencing and other functions, is considered by the industry to be the third generation of therapeutic drugs after chemical small molecule drugs and biological macromolecule drugs. This therapy achieves the treatment of diseases at the gene level and is not restricted by non-drugable proteins. As the most mainstream type of gene therapy, RNAi technology treats diseases at the mRNA level, which is more efficient than chemical small molecule drugs and biological macromolecule drugs at the protein level. Using RNAi technology, the sense and antisense strand sequences of siRNA with high specificity and good inhibitory effect can be designed according to specific gene sequences. These single-stranded sequences are synthesized through solid phase, and then the sense and antisense strands are paired into siRNA according to the base pairing principle in a specific annealing buffer, and finally delivered to the corresponding target in the body through a carrier system, degrading the target mRNA and destroying the function of the target mRNA as a translation template, thereby preventing the synthesis of related proteins.

siRNA delivery system

siRNA is unstable in blood and tissues and is easily degraded by nucleases. In order to improve the stability of siRNA, the sense strand and/or antisense strand of siRNA can be modified, but these chemical modifications only provide limited protection from nuclease degradation and may ultimately affect the activity of siRNA. Therefore, a corresponding delivery system is also needed to ensure that siRNA passes through the cell membrane safely and efficiently. Since siRNA has a large molecular weight, carries a large amount of negative charge, and has high water solubility, it cannot pass through the cell membrane smoothly and reach the cell.

The basic structure of liposomes is composed of a hydrophilic core and a phospholipid bilayer. It has a phospholipid bilayer similar to a biological membrane and has high biocompatibility. Therefore, liposomes once became the most popular and widely used siRNA carrier. Liposome-mediated siRNA delivery mainly encapsulates siRNA into liposomes to protect siRNA from degradation by nucleases, improve the efficiency of siRNA passing through cell membrane barriers, and thus promote cell absorption. For example, anionic liposomes, pH-sensitive liposomes, immunoliposomes, fusogenic liposomes, and cationic lipids, etc. Although certain progress has been made, liposomes themselves are prone to induce inflammatory reactions. Before administration, a variety of antihistamines and hormones such as cilitizine and dexamethasone must be used to reduce possible acute inflammatory reactions. Therefore, in actual clinical applications, it is not suitable for all treatment areas, especially in the treatment of some chronic diseases. The cumulative toxicity that may be caused by long-term use is a potential safety hazard. Therefore, a safer and more effective carrier system is needed to deliver siRNA.

The asialoglycoprotein receptor (ASGPR) in the liver is a receptor specifically expressed by hepatocytes and is a highly efficient endocytic receptor. Since the secondary end of various glycoproteins exposed after enzyme or acid hydrolysis of sialic acid in the body under physiological conditions is a galactose residue, the sugar that ASGPR specifically binds is galactosyl, so it is also called galactose-specific receptor. Monosaccharide and polysaccharide molecules such as galactose, galactosamine, and N-acetylgalactosamine have high affinity for ASGPR. The main physiological function of ASGPR is to mediate the clearance of asialoglycoproteins, lipoproteins and other substances in the blood, and it is closely related to the occurrence and development of liver diseases such as viral hepatitis, cirrhosis, and liver cancer. The discovery of this characteristic of ASGPR plays an important role in the diagnosis and treatment of hepatic diseases (Ashwell G, Harford J, Carbohydrate specific Receptors of the Liver, Ann Rev Biochem 1982 51:531-554). The therapeutic drugs for liver-derived diseases containing galactose or galactosamine and their derivatives in the structure can specifically bind to ASGPR, thereby having active liver targeting and requiring no other carrier system for delivery.

Complement system

The activation of the complement system includes three pathways: the classical pathway, the lectin pathway, and the alternative pathway. The main difference between these three pathways is the initiation process. After initiation, C3 convertase is formed and further cleaves C3. The C3b fragment combines with the previous complex to form C5 convertase, which cleaves C5b and C5a, causing the assembly of MAC, which cleaves cells and stimulates an inflammatory response to remove foreign substances.

The classical pathway (CP) is usually initiated by the attachment of C1q to an immune complex such as IgM or IgG. C1q then activates C1r, which changes the conformation of the C1r2-C1s2 structure. C1r releases C1s, which has serine protease (SP) activity and cleaves C4 and C2 to form C4b2a (C3 convertase). C3 convertase forms C5 convertase by cleaving C3 and subsequently binding to C3b. C4a, C3a, and C5a stimulate the inflammatory response. C5b forms C5b-9MAC with the help of C6, C7, C8, and C9 to attack host cells or pathogens.

Activation of the lectin pathway (LP) is identical to activation of the classical pathway (CP), except that the initiator of the LP is mannose-binding lectin (MBL), which forms a multimeric lectin complex by binding to ficolin. This binding leads to activation of the mbl-associated serine proteases (MASPs), which trigger the complement system. MASP-1 and MASP-2 are similar to C1r and C1s, respectively. MASP-1 and MASP-2 fully activate the complement system by cleaving C4 and C2 to form the C3 convertase.

The alternative activation pathway (AP) differs from the classical pathway (CP) in that activation bypasses the three components of C1, C4, and C2 and directly activates C3. When pathogen invasion is detected, a large number of C3 thioester domains (TED) become metastable, exposing C3 to the factor b (Fb) binding site. C3b is recognized by factor B to form the complex C3bB. The C3bB complex is in turn cleaved by factor D to produce the active form of C3 convertase (C3bBb). C3 convertase (C3bBb) breaks down C3 into C3a and C3b. C3b binds to pathogens or target cells as an active fragment. In response to C3 convertase, C3b produces C5 convertase, which cleaves C5 to recruit the membrane attack complex (MAC) to lyse pathogens. Both types of C3 convertase (C3bB\C3bBb) will cleave C3 to form C3b. C3b then binds more factor B to enhance complement activation through AP. Alternatively, C3b leads to the formation of active C5 convertase (C3bBbC3b or C4bC2bC3b), which cleaves C5 and triggers later events leading to the formation of the membrane attack complex (MAC) (C5b-9).

Diseases resulting from inappropriate activation of the complement system

Inhibitors that inhibit the expression of complement C3 and C5 genes can be used to treat diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, rheumatoid arthritis, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), schizophrenia, Parkinson's disease (PD), prion diseases, complement component C3-related eye diseases, ischemia-reperfusion injury and neurodegenerative diseases.

Inhibitors that inhibit the expression of complement factor B genes can be used to treat subjects with complement factor B-associated disorders, such as C3 glomerulopathy, systemic lupus erythematosus (SLE) such as lupus nephritis, IgA nephropathy, diabetic nephropathy, and polycystic kidney disease.

Combination of drugs

The complement inhibitors available on the market include Soliris, Ultomiris, Empaveli, Eculizumab, Ravulizumab or Iptacopan (LNP023). These peptides, monoclonal antibodies or small molecule drugs can be used in combination with RNAi inhibitors that inhibit the expression of the complement system, thereby improving the ability of the whole body to inhibit the complement system. However, most of the current complement inhibitors are evaluated in clinical practice and are administered once a week to once every two weeks. The cost is relatively high and they can also cause low-level hemolysis in PNH subjects. Therefore, patients with the complement system still need a new alternative drug or combination drug.

Summary of the invention

In one aspect, the present invention provides an RNA inhibitor or a pharmaceutically acceptable salt thereof for inhibiting the expression of complement system genes.

An RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, wherein the RNA inhibitor is formed by base pairing of a sense strand and an antisense strand with a chain length of 15-30, preferably 19-23, wherein the antisense strand includes a region complementary to an mRNA encoding a complement system, and wherein the complementary region comprises at least 15 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides from the antisense strand of any one of Tables 1-1, 1-2, and 1-3.

The aforementioned RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, wherein the RNA inhibitor is an RNA inhibitor for inhibiting CFB gene expression, wherein the antisense strand includes a region complementary to the target sequence, the target sequence is: 5'gucuagucaacuuaauugaga 3' SEQ ID NO: 25, the starting position in NM_001710.5 is at 1157, and there is at least 85% base complementarity between the sense strand and the antisense strand.

The aforementioned RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, wherein the RNA inhibitor is an RNA inhibitor for inhibiting C5 gene expression, wherein the antisense strand includes a region complementary to the target sequence, the target sequence is: 5'uugucccaguauucuauguuu 3'SEQ ID NO: 826, the starting position in NM_001735.3 is at position 3073, and there is at least 85% base complementarity between the sense strand and the antisense strand.

The aforementioned RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, wherein the RNA inhibitor is an RNA inhibitor for inhibiting C3 gene expression, wherein the antisense strand includes a region complementary to a target sequence, the target sequence is: 5'gguguugacagauacaucu 3' SEQ ID NO: 1952, the starting position in NM_000064.4 is at position 4329; 5'ggagccuacagagaaauucua 3' SEQ ID NO: 2039, the starting position in NM_000064.4 is at position 771; the target sequence is: 5'agaaa uucuacuacaucuaua 3'SEQ ID NO: 2048, the starting position in NM_000064.4 is 782; the target sequence is: 5'gcugaggagaauugcuucaua 3'SEQ ID NO: 2240, the starting position in NM_000064.4 is 4603; the target sequence is: 5'ggagaauugcuucauacaaaa 3'SEQ ID NO: 2245, the starting position in NM_000064.4 is 4608; there is at least 85% base complementarity between the sense chain and the antisense chain.

The aforementioned CFB RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the antisense strand is selected from the following sequences:

5'ucucaauuaaguugacuagacac 3'SEQ ID NO: 293;

or a sequence having at least 15 consecutive nucleotides identical to the antisense strand, or a sequence differing from the antisense strand by one, two or three nucleotides, the sense strand may be any sequence containing at least 85% base complementarity with the antisense strand;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide.

The aforementioned CFB RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is selected from the following sequences:

5'gucuagucaacuuaauugaga 3'SEQ ID NO: 25;

or a sequence having at least 15 consecutive nucleotides identical to the sense strand, or a sequence differing from the sense strand by one, two or three nucleotides, the sense strand being any sequence containing at least 85% base complementarity with the antisense strand;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine.

The aforementioned C5 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the antisense strand is selected from the following sequences:

5'aaacauagaauacugggacaacg 3'SEQ ID NO: 1278;

or a sequence having at least 15 consecutive nucleotides identical to the antisense strand, or a sequence differing from the antisense strand by one, two or three nucleotides, the sense strand may be any sequence containing at least 85% base complementarity with the antisense strand;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide.

The aforementioned C5 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is selected from the following sequences:

5'uugucccaguauucuauguuu 3'SEQ ID NO: 826;

or a sequence having at least 15 consecutive nucleotides identical to the sense strand, or a sequence differing from the sense strand by one, two or three nucleotides, the antisense strand may be any sequence containing at least 85% base complementarity with the sense strand;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine.

The aforementioned C3 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the antisense strand is selected from the following sequences:

5'agauguaucugucaacaccau 3'SEQ ID NO.2543;

5'uagaauuucucuguaggcuccac 3'SEQ ID NO.2630;

5'uauagauguaguagaauuucucu 3'SEQ ID NO.2639;

5'uaugaagcaauucuccagcac 3'SEQ ID NO.2831;

5'uuuuguaugaagcaauucuccuc 3'SEQ ID NO.2590;

or a sequence having at least 15 consecutive nucleotides identical to the antisense strand, or a sequence differing from the antisense strand by one, two or three nucleotides,

Wherein, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide; the sense strand can be any sequence that contains at least 85% base complementarity with the antisense strand.

The aforementioned C3 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is selected from the following sequences:

5'gguguugacagauacaucu 3'SEQ ID NO.1952;

5'ggagccuacagagaaauucua 3'SEQ ID NO.2039;

5'agaaauucuacuacaucuaua 3'SEQ ID NO.2048;

5'gcugaggagaauugcuucaua 3'SEQ ID NO.2240;

5'ggagaauugcuucauacaaaa 3'SEQ ID NO.2245;

or a sequence having at least 15 consecutive nucleotides identical to the sense strand, or a sequence differing from the sense strand by one, two or three nucleotides,

Wherein, g = guanylate, a = adenylate, u = uridylate, c = cytidine; the antisense strand can be any sequence that contains at least 85% base complementarity with the sense strand.

The aforementioned CFB RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is SEQ ID NO: 25 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 293 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides:

Sense strand: 5'gucuagucaacuuaauugaga 3'SEQ ID NO: 25;

Antisense strand: 5'ucucaauuaaguugacuagacac 3' SEQ ID NO: 293;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide.

The aforementioned C5 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is SEQ ID NO: 826 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 1278 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides:

Sense strand: 5'uugucccaguauucuauguuu 3' SEQ ID NO: 826;

Antisense strand: 5'aaacauagaauacugggacaacg 3'SEQ ID NO: 1278;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide.

The aforementioned C3 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is SEQ ID NO: 2048 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 2639 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides:

Sense strand: 5'agaaauucuacuacaucuaua 3' SEQ ID NO: 2048;

Antisense strand: 5'uauagauguaguagaauuucucu 3' SEQ ID NO: 2639;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide.

The aforementioned C3 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is SEQ ID NO: 2639 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 2831 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides:

Sense strand: 5'gcugaggagaauugcuucaua 3'SEQ ID NO: 2240;

Antisense strand: 5'uaugaagcaauucuccucagcac 3' SEQ ID NO: 2831;

Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide.

In the aforementioned RNA inhibitor or a pharmaceutically acceptable salt thereof, at least one nucleotide of the RNA inhibitor is modified.

The aforementioned RNA inhibitor or its pharmaceutically acceptable salt, the modification includes: 2'-fluoro modification, 2'-methoxy modification, thiophosphate modification, invAb modification, glycerol nucleotide, 3' terminal deoxythymine (dT) nucleotide, locked nucleotide, unlocked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, 2' phosphate modification or 2-O-(N-methylacetamide) modification, morpholino nucleotide, aminophosphoryl, abasic nucleotide, abasic deoxynucleotide, nucleotide containing non-natural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, methylphosphonate modification, 5'-phosphate modification, 5'-phosphate analog modification, thermally unstable nucleotide, nucleotide analogs one or a combination of several thereof.

The aforementioned CFB RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the antisense strand is selected from the following sequences:

5'UsdCsUCdAATUAAGTUfGACUAGACsAsC 3'SEQ ID NO: 588;

Among them, G＝2'-O-methylguanylate, A＝2'-O-methyladenylate, U＝2'-O-methyluridylate, C＝2'-O-methylcytidine; Gs＝2'-O-methyl-3'-thioguanylate, As＝2'-O-methyl-3'-thioadenylate, Cs＝2'-O-methyl-3'-thiocytidine; fG＝2'-fluoroguanylate, fA＝2'-fluoroadenylate, fU＝2'-fluorouridylate, fC＝2'-fluorocytidine; fGs＝2'-fluoro-3'-thioguanylate, T＝2'-deoxy-thymidylate, dA＝2'-deoxy-adenylate, dG＝2'-deoxy-guanylate; the sense strand can be any sequence containing at least 85% base complementarity with the antisense strand, and the modification method is not limited.

The aforementioned C5 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the antisense strand is selected from the following sequences:

5'AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3'SEQ ID NO: 1598;

Among them, G＝2'-O-methylguanylate, A＝2'-O-methyladenylate, U＝2'-O-methyluridylate, C＝2'-O-methylcytidine; Gs＝2'-O-methyl-3'-thioguanylate, As＝2'-O-methyl-3'-thioadenylate, Cs＝2'-O-methyl-3'-thiocytidine; fG＝2'-fluoroguanylate, fA＝2'-fluoroadenylate, fU＝2'-fluorouridylate, fC＝2'-fluorocytidine; fGs＝2'-fluoro-3'-thioguanylate, T＝2'-deoxy-thymidylate, dA＝2'-deoxy-adenylate, dG＝2'-deoxy-guanylate; the sense strand can be any sequence containing at least 85% base complementarity with the antisense strand, and the modification method is not limited.

The aforementioned C3 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the antisense strand is selected from the following sequences:

5'UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3'SEQ ID NO: 1665;

5'UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'SEQ ID NO: 1671;

Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-fluorouridine Acid, fC＝2'-fluorocytidylic acid; fGs＝2'-fluoro-3'-thioguanylic acid, fAs＝2'-fluoro-3'-thioadenylic acid, fUs＝2'-fluoro-3'-thiouridylic acid, fCs＝2'-fluoro-3'-thiocytidylic acid, T＝2'-deoxy-thymidylic acid, dG＝2'-deoxy-guanylic acid, dAs＝2'-deoxy-3'-thioadenylic acid; the sense strand can be any sequence containing at least 85% base complementarity with the antisense strand, and the modification method is not limited.

The aforementioned CFB RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is selected from the following sequences:

5’GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3’SEQ ID NO: 559;

Among them, G＝2'-O-methylguanylic acid, A＝2'-O-methyladenylic acid, U＝2'-O-methyluridylic acid, C＝2'-O-methylcytidylic acid; Gs＝2'-O-methyl-3'-thioguanylic acid, As＝2'-O-methyl-3'-thioadenylic acid, Us＝2'-O-methyl-3'-thiouridylic acid, Cs＝2'-O-methyl-3'-thiocytidylic acid; fG＝2'-fluoroguanylic acid, fA＝2'-fluoroadenylic acid, fU＝2'-fluorouridylic acid, fC＝2'-fluorocytidylic acid; the antisense strand can be any sequence containing at least 85% base complementarity with the sense strand, and the modification method is not limited.

The aforementioned C5 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is selected from the following sequences:

5’UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3’SEQ ID NO: 1537;

Among them, G＝2'-O-methylguanylic acid, A＝2'-O-methyladenylic acid, U＝2'-O-methyluridylic acid, C＝2'-O-methylcytidylic acid; Gs＝2'-O-methyl-3'-thioguanylic acid, As＝2'-O-methyl-3'-thioadenylic acid, Us＝2'-O-methyl-3'-thiouridylic acid, Cs＝2'-O-methyl-3'-thiocytidylic acid; fG＝2'-fluoroguanylic acid, fA＝2'-fluoroadenylic acid, fU＝2'-fluorouridylic acid, fC＝2'-fluorocytidylic acid; the antisense strand can be any sequence containing at least 85% base complementarity with the sense strand, and the modification method is not limited.

The aforementioned C3 RNA inhibitor or a pharmaceutically acceptable salt thereof, wherein the sense strand is selected from the following sequences:

5'AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3'SEQ ID NO: 1645;

5’GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3’SEQ ID NO: 1651;

Among them, G＝2'-O-methylguanylic acid, A＝2'-O-methyladenylic acid, U＝2'-O-methyluridylic acid, C＝2'-O-methylcytidylic acid; Gs＝2'-O-methyl-3'-thioguanylic acid, As＝2'-O-methyl-3'-thioadenylic acid, Us＝2'-O-methyl-3'-thiouridylic acid, Cs＝2'-O-methyl-3'-thiocytidylic acid; fG＝2'-fluoroguanylic acid, fA＝2'-fluoroadenylic acid, fU＝2'-fluorouridylic acid, fC＝2'-fluorocytidylic acid; the antisense strand can be any sequence containing at least 85% base complementarity with the sense strand, and the modification method is not limited.

As a preferred embodiment, the aforementioned CFB RNA inhibitor or a pharmaceutically acceptable salt thereof, the sense strand is SEQ ID NO: 559 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 588 or a sequence that differs therefrom by one, two or three nucleotides:

Sense strand: 5’GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3’SEQ ID NO: 559;

Antisense strand: 5'UsdCsUCdAATUAAGTUfGACUAGACsAsC 3'SEQ ID NO: 588;

Wherein, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, f A = 2'-fluoroadenylic acid, fU = 2'-fluorouridylic acid, fC = 2'-fluorocytidylic acid; fGs = 2'-fluoro-3'-thioguanylic acid, fAs = 2'-fluoro-3'-thioadenylic acid, fUs = 2'-fluoro-3'-thiouridylic acid, fCs = 2'-fluoro-3'-thiocytidylic acid, T = 2'-deoxy-thymidylic acid, Ts = 2'-deoxy-3'-thiothymidylic acid, dA = 2'-deoxy-adenylic acid.

As a preferred embodiment, the aforementioned C5 RNA inhibitor or a pharmaceutically acceptable salt thereof, the sense strand is SEQ ID NO: 1537 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 1598 or a sequence that differs therefrom by one, two or three nucleotides:

Sense strand: 5’UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3’SEQ ID NO: 1537;

Antisense strand: 5'AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3'SEQ ID NO: 1598;

Wherein, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, f A = 2'-fluoroadenylic acid, fU = 2'-fluorouridylic acid, fC = 2'-fluorocytidylic acid; fGs = 2'-fluoro-3'-thioguanylic acid, fAs = 2'-fluoro-3'-thioadenylic acid, fUs = 2'-fluoro-3'-thiouridylic acid, fCs = 2'-fluoro-3'-thiocytidylic acid, T = 2'-deoxy-thymidylic acid, Ts = 2'-deoxy-3'-thiothymidylic acid, dA = 2'-deoxy-adenylic acid.

As a preferred embodiment, the aforementioned C3 RNA inhibitor or a pharmaceutically acceptable salt thereof, the sense strand is SEQ ID NO: 1651 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 1671 or a sequence that differs therefrom by one, two or three nucleotides:

Sense strand: 5’AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3’SEQ ID NO: 1645;

Antisense strand: 5'UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3'SEQ ID NO: 1665;

Alternatively, the sense strand is SEQ ID NO: 1645 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 1665 or a sequence that differs therefrom by one, two or three nucleotides:

Sense strand: 5'GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3'SEQ ID NO: 1651;

Antisense strand: 5'UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3'SEQ ID NO: 1671;

Wherein, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, f A = 2'-fluoroadenylic acid, fU = 2'-fluorouridylic acid, fC = 2'-fluorocytidylic acid; fGs = 2'-fluoro-3'-thioguanylic acid, fAs = 2'-fluoro-3'-thioadenylic acid, fUs = 2'-fluoro-3'-thiouridylic acid, fCs = 2'-fluoro-3'-thiocytidylic acid, T = 2'-deoxy-thymidylic acid, dG = 2'-deoxy-guanylic acid, dAs = 2'-deoxy-3'-thioadenylic acid.

In some embodiments, mismatches can be accommodated in the sense strand or antisense strand of the RNA inhibitor of the present invention. The mismatch position can occur at the 5' or 3' end or within the sequence. As a preferred embodiment, the mismatch is no more than 3 nucleotides, for example, 0, 1, 2, or 3 nucleotides.

In the technical method, preferably, the RNA inhibitor of the present invention or a pharmaceutically acceptable salt thereof further contains carrier structures 5'MVIP and 3'MVIP, and the structure of the RNA inhibitor is as shown in Formula Ia, Ib or Ic:

in,

The vector structure includes 5'MVIP (5'MultiValent Import Platform) and 3'MVIP (3'MultiValent Import Platform);

5'MVIP consists of a transfer point R ₁ , a connecting chain D, a linker B, a branch chain L and a liver-targeting specific ligand X, which is connected to the 5' end of the sense chain or the 5' end of the antisense chain through the transfer point R _1. Its structure is shown in general formula I:

(XL) _n -BDR ₁ -

I

3'MVIP consists of a transfer point R ₂ , a connecting chain D, a linker B, a side chain L and a liver-targeting specific ligand X, which is connected to the 3' end of the sense chain or the 3' end of the antisense chain through the transfer point R _2. Its structure is shown in general formula II:

(XL) _m -BDR ₂ -

II

in,

n and m are each independently any integer of 0-4, each independently preferably an integer of 1-3, and n+m=an integer of 2-6, preferably n+m=2, 3 or 4, more preferably 4;

The transition point _R1 is a heterocyclic or carbocyclic structure containing N, S or O as shown below:

Alternatively, R ₁ is -NH(CH ₂ ) _x CH ₂ O-, wherein x is any integer from 3 to 12, preferably any integer from 4 to 6;

The transition point _R2 is a heterocyclic or carbocyclic structure containing N, S or O as shown below:

Alternatively, the transition point R ₂ is -NH(CH ₂ ) _x1 CH(OH)(CH ₂ ) _x2 CH ₂ O-, wherein x1 is any integer from 1 to 4, and x2 is any integer from 0 to 4;

The liver-targeting specific ligand X is selected from the structure used to enhance the uptake of RNA inhibitors by hepatocytes, and is the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP, and is selected from monosaccharides and their derivatives, preferably N-acetylgalactosamine and its derivatives, and more preferably selected from the following structures:

Wherein, W is selected from one or two of -OH, -NHCOOH and -NHCO(CH ₂ ) _q CH ₃ , wherein q is an integer of 0-4;

The branched chain L is the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP, and is selected from one or more of the following structures:

wherein r1 is any integer from 1 to 12, r2 is any integer from 0 to 20, and Z is H, an alkyl group or an amide group, and the alkyl group is, for example, a C ₁ -C ₅ alkyl group;

The linker B is the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP, and is selected from the following structures:

wherein _A1 and _A2 are each independently C, O, S, -NH-, carbonyl, amide, phosphoryl or thiophosphoryl, and r is any integer from 0 to 4;

The connecting chain D is the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP, and is selected from the following structures:

Wherein, each p is independently any integer of 1-20; s is any integer of 2-13; _Z1 and _Z2 are the same or different substituent groups, such as _C3 - _C10 alkyl.

In some embodiments, 5'MVIP is selected from any one of 5'MVIP01 to 5'MVIP22 in Table 11.

In some embodiments, the 3’MVIP is selected from any one of 3’MVIP01 to 3’MVIP27 in Table 12.

In some embodiments, in the RNA inhibitor of the present invention or a pharmaceutically acceptable salt thereof, 5'MVIP is 5'MVIP01 or 5'MVIP09 as shown below, and 3'MVIP is 3'MVIP01, 3'MVIP09 or 3'MVIP17 as shown below:

In some embodiments, the RNA inhibitor of the present invention or a pharmaceutically acceptable salt thereof, wherein the combination of the sense chain 5’MVIP and the antisense chain 3’MVIP is 5’MVIP01/3’MVIP01, 5’MVIP01/3’MVIP17 or 5’MVIP09/3’MVIP09, or the combination of the sense chain 5’MVIP and the sense chain 3’MVIP is 5’MVIP01/3'MVIP09 or 5’MVIP09/3’MVIP01.

On the other hand, the CFB RNA inhibitor is selected from Kylo-17-DS2911.

On the other hand, the C5 RNA inhibitor is selected from Kylo-19-DS7881.

On the other hand, the C3 RNA inhibitor is selected from Kylo-27-DS8201 and Kylo-27-DS8141.

In another aspect, the RNA inhibitor of the present invention or a pharmaceutically acceptable salt thereof is used in the preparation of a medicament for treating and/or preventing diseases associated with increased levels of the complement system, including but not limited to lipid metabolism disorders.

In another aspect, a pharmaceutical composition comprises the aforementioned RNA inhibitor or other therapeutic agents for treating or preventing complement system-related diseases.

On the other hand, a pharmaceutical composition comprises the aforementioned RNA inhibitor or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, and the dosage form is an oral agent, an intravenous injection, or a subcutaneous or intramuscular injection, preferably a subcutaneous injection.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below.

The accompanying drawings are briefly described as follows:

FIG1 is a high-resolution mass spectrum of ERCd-01-c2 synthesized in 3.1.15 in Example 3 of the present application;

FIG2 is a high-resolution mass spectrum of 3'MVIP17-c1 synthesized in 3.1.2.6 of Example 3 of the present application;

FIG3 is a high-resolution mass spectrum of 5'MVIP09-ERCd-PFP-c2 synthesized in 3.2.1.2 of Example 3 of the present application;

FIG4 is a schematic diagram showing the effect of hCFB administration of the present invention on the inhibition rate of hCFB protein in mouse serum;

FIG5 is a schematic diagram showing the effect of hC5 administration of Example 7-2 of the present invention on the inhibition rate of hC5 protein in mouse serum;

6 is a schematic diagram showing the effect of hC5 administration of Example 7-3 of the present invention on the inhibition rate of hC5 protein in mouse serum;

7 is a schematic diagram showing the effect of hC3 administration of the present invention on the inhibition rate of hC3 protein in mouse serum;

FIG8 is a schematic diagram showing the effect of the CFB RNA inhibitor of the present invention on the inhibition rate of CFB protein in NHP cynomolgus monkeys;

Figure 9 is a schematic diagram showing the effect of the CFB RNA inhibitor of the present invention on the CFB mRNA level in the liver tissue of cynomolgus monkeys;

Figure 10 is a schematic diagram showing the effect of the C5 RNA inhibitor of the present invention on the inhibition rate of C5 protein in NHP cynomolgus monkeys;

Figure 11 is a schematic diagram showing the effect of the C5 RNA inhibitor of the present invention on the retention level of CH50 activity in NHP cynomolgus monkeys;

Figure 12 is a schematic diagram showing the effect of the C3 RNA inhibitor of the present invention on the inhibition rate of C3 protein in NHP cynomolgus monkeys;

DETAILED DESCRIPTION

The following is an explanation of the implementation of the present invention by means of specific embodiments. A person skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

Definition of terms

In the present application, examples of complement system mRNA sequences are readily obtained using publicly available databases, such as GenBank, UniProt, OMIM and the Macaca Genome Project website.

The term "CFB" refers to complement factor B, whose mRNA sequences can be found, for example, in GenBank NM_001710.5; 001710; cynomolgus monkey NC_041757.1, mouse NM_001142706 and NM_008198; rat NM_212466.3.

The term "C3" refers to complement C3, whose mRNA sequence can be found, for example, in GenBank NM_000064.4; cynomolgus monkey NC_000019.10; mouse NM_009778; rat NM_016994.

The term "C5" refers to complement C5, whose mRNA sequence can be found, for example, in GenBank NM_001735.3; mouse NM_013485; rat NM_057146.

Indicators for judging "inhibiting the expression of CFB" include: inhibition of the mRNA level of the CFB gene, inhibition of the protein level of the CFB gene, and any other level of inhibition; and may also be: CH50 activity as a measure of total hemolytic complement, AH50 for measuring the hemolytic activity of the complement alternative pathway, and/or lactate dehydrogenase (LDH) level as a measure of intravascular hemolysis, and/or hemoglobin level; the levels of C3, C9, C5, C5a, C5b and soluble C5b-9 complex may also be measured to evaluate the CFB expression level.

The judgment indicators of "inhibition of C3 expression" include: C3 determination, total complement determination, immune complex determination, C3 plasma concentration determination, and C3 fixed antibody method test; C3 determination can be any level of inhibition, such as inhibition of the mRNA level of the C3 gene, or inhibition of the protein level of the C3 gene; C3 determination may be performed together with other complement components to evaluate the function of the entire complement system. This usually includes measuring C4 concentration and indicators such as CH50; immune complex determination: immune complex is a structure composed of antibodies and antigens, which is formed in certain autoimmune diseases. Serum immune complex determination can be used to detect these immune complexes to evaluate whether they cause activation of the immune system, leading to a decrease in C3; C3 plasma concentration determination: evaluate the function of the complement system by measuring the concentration of C3 in plasma. Under normal circumstances, the concentration of C3 should be within the normal range; C3 fixed antibody method: This method is used to measure the ability of C3 to bind to specific antigens to evaluate the function of the complement system. It can also be to measure indices related to C3 gene levels such as CH50 activity as a measure of total hemolytic complement, AH50 to measure the hemolytic activity of the alternative complement pathway, and/or lactate dehydrogenase (LDH) levels as a measure of intravascular hemolysis, and/or hemoglobin levels. C3 expression can also be evaluated by measuring the levels of CFB, C9, C5, C5a, C5b, and soluble C5b-9 complexes.

Indicators for judging "inhibiting the expression of C5" include: inhibition of the mRNA level of the C5 gene, inhibition of the protein level of the C5 gene, and any other level of inhibition; it can also be: CH50 activity as a measure of total hemolytic complement, AH50 for measuring the hemolytic activity of the complement alternative pathway, and/or lactate dehydrogenase (LDH) level as a measure of intravascular hemolysis, and/or hemoglobin level; the levels of C3, C9, C5, C5a, C5b and soluble C5b-9 complex can also be measured to evaluate the CFB expression level.

In the present application, "target sequence" refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the complement system gene, including mRNA that is the product of RNA processing of the primary transcription product. In some embodiments, the target portion of the sequence will be at least long enough to be used as a substrate for RNA inhibitor-guided degradation at or near the portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the complement system gene. The length of the "target sequence" is generally about 15-30 nucleotides.

In the present application, the term "RNA inhibitor" generally refers to an agent comprising RNA as defined in the present term, and which can mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. Directing sequence-specific degradation of mRNA via a process known as RNA inhibition, regulating (e.g., inhibiting) expression of complement system genes in cells (e.g., cells in a subject such as a mammalian subject).

In some embodiments, the RNA inhibitor can be a single-stranded siRNA (ssRNA inhibitor) introduced into a cell or organism to inhibit a target mRNA (i.e., a complement system gene). The single-stranded RNA inhibitor binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides in length and are chemically modified.

In some embodiments, the "RNA inhibitor" used in the present application is a double-stranded RNA, and is referred to as a "double-stranded RNA inhibitor", "double-stranded RNA (dsRNA, DS) molecule", "dsRNA agent" or "dsRNA" in the present invention. The term "dsRNA" refers to a complex of ribonucleic acid molecules, which has a duplex structure comprising two antiparallel and substantially complementary nucleic acid chains, referred to as having "sense" and "antisense" orientations relative to the target mRNA. In some embodiments of the present application, double-stranded RNA (dsRNA) triggers the degradation of the target mRNA through a post-transcriptional gene silencing mechanism (referred to as RNA inhibition or RNA interference in the present invention).

The duplex structure can be any length that triggers the specific degradation of complement system mRNA through the RISC pathway, and can be in the range of about 15 to 36 base pairs, for example, about 15-30 base pairs in length, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 36 base pairs in length. In certain embodiments, the RNA inhibitor of the present application is a dsRNA of 15-30 nucleotides that interacts with the target sequence to guide the cutting of the complement system mRNA.

Typically, most of the nucleotides in the sense strand and antisense strand of a dsRNA molecule are ribonucleotides, but as described in detail in the present invention, one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides, may also be included. In addition, the RNA inhibitors involved in this specification may include chemically modified ribonucleotides, and may have modified nucleotides in multiple regions. The term "modified nucleotides" used in the present invention means independently having a modified sugar moiety, a modified internucleotide connection or a modified nucleobase, or a nucleotide in any combination thereof. Therefore, the term "modified nucleotides" encompasses replacement, addition or removal of, for example, functional groups or atoms of internucleotide connections, sugar moieties or nucleobases. Modifications applicable to the RNA inhibitors of the present application include all types of modifications disclosed in the present invention or known in the art.

In this application, the term "nucleotide sequence" generally refers to a series or a certain order of nucleotides, whether modified or unmodified, using standard nucleotide nomenclature and the symbol table of modified nucleotides described in this application as a series of letters. The nucleotide sequence described in this application is a polymer composed of phosphodiester bonds (or its related structural variants or synthetic analogs), including naturally occurring nucleotide polymers, but it should be understood that the scope of the term also includes various analogs, including but not limited to: peptide nucleic acids (PNA), aminophosphoroesters, thiophosphates, methylphosphonates and 2'-O-methyl ribonucleic acids, etc. There are usually about 15-30 nucleotides, but the term can also refer to molecules of any length.

In some embodiments, the nucleotide sequence comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleotides. The term "modified nucleotide sequence" generally means a series or a certain order of nucleotides comprising at least one modification and/or at least one modified internucleotide linkage.

In the present application, the term "modified nucleotides" generally means comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleotides. For example, 2'-deoxy-thymidylic acid 2'-O-methyl modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-long chain alkyl-modified nucleotides (such as hexadecyl), morpholino nucleotides, phosphoramidate nucleotides, non-natural core base nucleotides, 5'-phosphorothioate nucleotides, and nucleotides connected with cholesterol derivatives or dodecanoic acid didecylamide groups.

Modified nucleotides contain modified sugar groups and/or modified nucleobases.

In this application, the term "nucleobase" or "base" generally refers to a heterocyclic pyrimidine or purine compound, which is a component of all nucleic acids and includes adenine, guanine, cytosine, thymine and uracil. Nucleotides can include modified nucleotides or nucleotide mimetics, abasics or surrogate replacement parts. The term "unmodified nucleobase" or "naturally occurring nucleobase" generally refers to the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine and guanine; and the pyrimidine bases thymine, cytosine and uracil. "Modified nucleobase" generally refers to any nucleobase that is not a naturally occurring nucleobase.

In this application, the term "sugar" generally refers to a naturally occurring sugar or a modified sugar of a nucleotide. The term "naturally occurring sugar" generally refers to a furanose ribosyl as found in naturally occurring RNA or a deoxyfuranose ribosyl as found in naturally occurring DNA. "Modified sugar" refers to a substituted sugar or sugar surrogate, for example, a fluoro or methoxy substitution at the 2' position of the sugar.

In this application, the term "internucleotide linkage" generally means a covalent linkage between adjacent nucleotides in a nucleotide sequence. "Naturally occurring internucleotide linkage" means a 3' to 5' phosphodiester linkage. "Modified internucleotide linkage" means any internucleotide linkage other than a naturally occurring internucleotide linkage.

In the present application, the term "antisense strand" (AS) generally refers to a strand of an RNA inhibitor (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence. When used in the present invention, the term "region of complementarity" generally refers to a region on the antisense strand that is substantially complementary to a sequence defined in the present application (e.g., a target sequence).

In the present application, the term "sense strand" (SS) generally refers to a strand of an RNA inhibitor (e.g., dsRNA) that includes a region that is substantially complementary to the region of an "antisense strand" (AS). The "sense" strand is sometimes referred to as a "sense" strand, a "passenger" strand, or an "anti-guide" strand. With the sequence of the sense strand, the antisense strand targets the desired mRNA, while the sense strand may target different targets or be degraded. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. The incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications or using 5' end caps on the sense strand.

The loading of the siRNA double strand into the ago protein must first be initiated by the recognition of the 5' end of the antisense strand, which is a prerequisite for placing the rest of the double strand into the AGO protein and nucleic acid binding channel. The MID domain of the AGO protein recognizes the nucleotide at the 5' end. After being recognized, the siRNA double strand is loaded into the AGO protein and nucleic acid binding channel to form pre-RISC. After the siRNA double strand is loaded into the nucleic acid binding channel, pre-RISC discharges the sense strand (passenger strand) and forms RISC with the remaining antisense strand (guide strand).

In this application, the term "complementary" refers to the ability of two nucleotide sequences to hybridize under certain conditions, form base pair hydrogen bonds, and form a duplex or double helix structure. For example, the antisense strand of an RNA inhibitor hybridizes with the sense strand of an RNA inhibitor or the complement system mRNA to form Watson-Crick base pairs or non-Watson-Crick base pairs, and includes natural or modified nucleotides or nucleotide mimetics. "Complementary" does not necessarily have nucleobase complementarity on each nucleoside. On the contrary, some mismatches can be tolerated.

In the present application, the term "mismatch" refers to when the complementary region is not completely complementary to the target sequence, and the mismatch can be in the core region or the terminal region. Usually, the most tolerated mismatch is in the terminal region, for example, within 5, 4, 3 or 2 nucleotides of the 5' end and/or the 3' end, and no more than 3 mismatches.

For example, the results of Gu S, Jin L, Zhang F, Huang Y, Grimm D, Rossi JJ, Kay MA. Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes. Proc Natl Acad Sci USA 2011, 108: 9208-9213. indicate that any factors that affect the thermodynamic stability of the double helix, such as mismatches and non-Watson-Crick base pairs, are conducive to the pre-RISC expelling the sense chain to form RISC.

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

In the present application, the term "pharmaceutically acceptable" generally refers to one or more non-toxic substances that do not inhibit the effectiveness of the biological activity of the active ingredient. Such preparations may generally contain salts, excipients, buffers, preservatives, compatible carriers and optional other therapeutic agents. Such pharmaceutically acceptable preparations may also generally contain compatible solid or liquid fillers, diluents or encapsulation materials suitable for administration to people. When used in medicine, salts should be pharmaceutically acceptable salts, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts, and they cannot be excluded from the scope of this application. Such pharmacological and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts may also be prepared into alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

In the present application, the term "lipid nanoparticle" or "LNP" generally refers to a vesicle comprising a lipid layer encapsulating a pharmacologically active molecule (e.g., dsRNA). LNP is described in, for example, Chinese Patent No. CN103189057B, the entire contents of which are incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides an RNA inhibitor or a pharmaceutically acceptable salt thereof for inhibiting the expression of complement system genes.

siRNA design

A group of siRNAs targeting human complement system genes were screened using self-designed sequence screening software: CFB human: GenBank NM_001710.5; C3 human: GenBank NM_000064.4; C5 human: GenBank NM_001735.3.

The primary sequence selected by CFB is shown in Table 1-1, and the primary sequence selected by C5 is shown in Table 1-2:

The modified secondary sequences selected by CFB are shown in Table 2-1, the secondary sequences selected by C5 are shown in Table 2-2, and the secondary sequences selected by C3 are shown in Table 2-3:

siRNA Synthesis siRNA was synthesized and annealed using conventional methods known in the art.

In certain embodiments, the duplex structure (complementary region) formed by the antisense strand and the sense strand comprises at least 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides.

Table 1-1 CFB primary sequence

Table 1-2 C5 primary sequence

Table 1-3 C3 primary sequence

Wherein n is a or u or g or c, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide.

In some screening embodiments, the sense strand and antisense strand of the RNAi agent are selected from the sequences in Table 1 or differ from each sequence in Table 1 by one, two or three nucleotides.

In some embodiments, the base pairs of the sense strand in Table 1 and the corresponding antisense strand in Table 1 are complementary to form dsRNA, which may be partially complementary or completely complementary. The partial complementarity may be at least 85% base pairing.

In some embodiments, the combination of the sense strand and the antisense strand is not limited to the double-strand combination in Table 1, and one of the sense strands in Table 1 can be complementary to any antisense strand.

The present invention aims to protect the core sequence of the sequence in Table 1, and the core sequence is any segment of the above sequence that is at least 15 consecutive nucleotides, wherein at least 15 refers to 15, 16, 17, 18, 19, 20, 21, 22, 23, etc. In some embodiments, the sense strand is as shown in formula (1): 5'-X core sequence Y-3', the antisense strand and the sense strand have at least 85% base complementarity, X and Y contain 0, 1, 2, 3, 4, 5, 6 nucleotides, and 0, 1, 2, 3 unpaired bases can be allowed to exist at the terminal position in the double strand.

The antisense strand contains consecutive nucleotides that differ from formula (2) by 0, 1, 2 or 3 nucleotides, formula (2): 5′-X′ core sequence Y′-3′, the antisense strand and the sense strand have at least 85% base complementarity, X′ and Y′ contain 0, 1, 2, 3, 4, 5, or 6 nucleotides, and 0, 1, 2, or 3 unpaired bases can be allowed to exist at the terminal positions in the double helix.

As an example, the core sequence allows 0, 1, 2, or 3 nucleotide differences, which may be base pairs formed according to the Watson-Crick principle or mismatches.

In some embodiments, the RNA inhibitor can be added to the cell line for sequence screening by cell transfection or liposome-nucleic acid nanoparticles, which are well known to those skilled in the art. Patents US9233971B2, US9080186B2, CN102985548B and CN103189057B on lipid compounds and methods for preparing liposome-nucleic acid nanoparticles are fully introduced into this specification.

In some embodiments, the amphoteric lipids in the lipid compound are preferably macrocyclic lipid compounds D1C1, T1C1, T1C6, T4C4, B2C1, B2C6, B2C7 and M10C1.

It is well known to those skilled in the art that dsRNAs having a duplex structure of about 20 to 23 base pairs, for example, 21 base pairs, have been found to be particularly effective in inducing RNA inhibition (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that shorter or longer RNA duplex structures are also effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). It is reasonable to expect that a duplex with a few nucleotides minus or added at one or both ends of a sequence in Tables 1 and 2 can be similarly effective compared to the dsRNA. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, 21 or more consecutive nucleotides derived from a sequence in Tables 1 and 2 and differing by no more than about 5, 10, 15, 20, 25 or 30% in their ability to inhibit complement system gene expression from a dsRNA comprising the entire sequence are included within the scope of the present application.

The dsRNA described in the present application may further include one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides. The nucleotide overhangs may include nucleotides/nucleoside analogs or combinations thereof, including deoxynucleotides. The overhangs may be on the sense strand, the antisense strand or a combination thereof. In addition, the nucleotides of the overhangs may be present at the 5' end, 3' end or both ends of the antisense strand or sense strand of the dsRNA. The overhangs may be formed by one strand being longer than the other strand, or by two strands of the same length being staggered. When the overhangs are in the antisense strand and may form mismatches or complementarity with the complement system mRNA or may be another sequence. For example, the overhangs are located at the 3' end of the sense strand, or alternatively, at the 3' end of the antisense strand.

The dsRNA may also have a blunt end, which means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhangs. The blunt end may be located at the 5' end of the antisense strand and the 3' end of the sense strand, or vice versa, or a double-ended blunt end, which is a double-stranded dsRNA over its entire length, i.e., there are no nucleotide overhangs at either end of the molecule.

In some embodiments, the sense strand or antisense strand of the dsRNA has a nucleotide overhang at the 3' end, the overhang contains 1, 2, 3 or 4 nucleotides, and the 5' end is blunt.

In some embodiments, the overhang is present at the 3' end of both the sense strand and the antisense strand, and the overhang contains 1, 2, 3 or 4 nucleotides, including but not limited to: TT, UU, AU or UA.

In some embodiments, the dsRNA is a double-ended blunt-ended dsRNA of 19, 21, or 23 nucleotides in length, which is double-stranded throughout its entire length, ie, there are no nucleotide overhangs at either end of the molecule.

In some embodiments, the dsRNA is 21 nucleotides in length, and both the sense and antisense strands have 2 nucleotide overhangs at the 3' end.

In order to enhance the in vivo stability of the RNA inhibitor described in the present application, the sense strand and antisense strand of the RNA inhibitor may be modified without affecting its activity or even enhancing its activity, wherein the nucleotides may have a modifying group, and the entire strand or a portion thereof may be modified. In some embodiments, one or more nucleotides on the sense strand and/or antisense strand are modified to form modified nucleotides.

In some embodiments, the sense strand and antisense strand of the RNA inhibitor (e.g., dsRNA) described herein are unmodified. In other embodiments, the sense strand and antisense strand of the RNA inhibitor described herein are chemically modified or coupled as known in the art and as described herein to enhance stability or other favorable properties. In other embodiments of the application, all or substantially all nucleotides of the RNA inhibitor described herein may be modified, i.e., the strand of the RNA inhibitor has no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

The sense strand and antisense strand of the RNA inhibitor as described in the present application can be synthesized and/or modified by methods known in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated by reference into the present invention. In the RNA inhibitor provided in the present application, the sense strand and antisense strand of the RNA inhibitor do not need to be uniformly modified, and one or more modifications can be incorporated into their single nucleotides.

In some embodiments, nucleotide modifications include: 5' terminal modified nucleotides, 3' terminal modified nucleotides, base modifications, sugar modifications or sugar substitutions, and backbone modifications. 5' terminal modifications refer to phosphorylation, coupling, and reverse connection. 3' terminal modifications refer to coupling, DNA nucleotides, reverse connection, etc.; base modifications refer to: replacement with stable bases, destabilizing bases, or bases that pair with an extended partner library, removal of bases (basic nucleotides), or conjugated bases. Sugar modifications are generally at the 2' position or the 4' position. Main chain modifications refer to: modification or substitution of phosphodiester bonds.

Specific nucleotide modifications may include, but are not limited to, 5'-terminal phosphorus-containing nucleotide modifications, vinylphosphonate deoxyribonucleotides, vinylphosphonate-containing nucleotides and cyclopropylphosphonate-containing nucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2-O-(N-methylacetamide) modified nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-F-arabino nucleotides, 5'-Me/2'-fluoro nucleotides, locked nucleotides, unlocked nucleotides, unlocked nucleobase analogs, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexanol-modified nucleotides, cyclohexenyl-modified nucleotides, methylphosphonate-containing nucleotides, thermally unstable nucleotides, GNA, deoxyribonucleotides, nucleotide analogs, morpholino nucleotides, abasic nucleotides, 3' to 3' linked (inverted) nucleotides, bridged nucleotides, peptide nucleic acids (PNAs).

The 5'-terminal phosphorus-containing group modified nucleotide can be a 5'-phosphate nucleotide or a nucleotide deoxynucleotide containing a 5'-phosphate analog; having, but not limited to: 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5-'-terminal phosphorothioate diester (5'-PS2), 5'-terminal vinyl phosphonate (5'-VP), 5'-terminal methyl phosphonate (MePhos) or 5'-deoxy-5'-C-malonyl. When the 5'-terminal phosphorus-containing group is 5'-terminal vinyl phosphonate (5'-VP), 5'-VP can be a 5'-E-VP isomer (i.e., trans-vinyl phosphonate), a 5'-Z-VP isomer (i.e., cis-vinyl phosphate) or a mixture thereof.

Modifications between nucleotides may include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates, phosphinates, phosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, boranephosphates, and also include various salts and free acids.

The end modification of the sense strand or antisense strand can avoid exonuclease degradation and enhance nuclease stability, such as the cap structure: inverted deoxy abasic cap (inverted deoxy abasic cap), abbreviated as invAb. invAb is well known in the art, and the specific performance verification can be found in F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16.

In some embodiments, the 2' position of the nucleotide sugar moiety at at least two or more even-numbered positions starting from the 5' end of the antisense strand is fluorine.

In some embodiments, all 2' positions of the nucleotide sugars at the even-numbered positions starting from the 5' end of the antisense strand are fluorine.

In some embodiments, at least one of the 2' positions of the nucleotide sugars at positions 2, 4, 6, 8, 12, and 14 starting from the 5' end of the antisense strand is fluorine. For example, the 2' positions of the nucleotide sugars at positions 2, 4, 6, 8, 12, and 14 starting from the 5' end of the antisense strand are all fluorine.

In some embodiments, except for the 2nd, 6th, 8th, 10th, 14th, and 16th nucleotides starting from the 5' end of the antisense strand, at least one of the 2' positions of the remaining nucleotide sugar groups is a methoxy group.

In some embodiments, except for the 2nd, 4th, 6th, 8th, 14th, and 16th nucleotides starting from the 5' end of the antisense strand, at least one of the 2' positions of the remaining nucleotide sugar groups is a methoxy group.

In some embodiments, except for the 2nd, 4th, 6th, 8th, 14th, 16th, 18th, and 20th nucleotides starting from the 5' end of the antisense strand, at least one of the 2' positions of the remaining nucleotide sugar groups is a methoxy group.

In some embodiments, at least two or more nucleotide sugar groups at odd-numbered positions starting from the 5' end of the sense strand have fluorine at the 2' position.

In some embodiments, the 2' positions of the nucleotide sugars at the odd-numbered positions starting from the 5' end of the sense strand are all fluorine.

In some embodiments, at least one of the 2' positions of the 5th, 7th, 8th, and 9th nucleotide sugars starting from the 5' end of the sense strand is fluorine. For example, the 2' positions of the 5th, 7th, 8th, and 9th nucleotide sugars starting from the 5' end of the sense strand are all fluorine.

In some embodiments, except for the 5th, 7th, 8th, and 9th nucleotides starting from the 5' end of the sense strand, at least one of the 2' positions of the remaining nucleotide sugar groups is a methoxy group.

In some embodiments, at least one of the 2' positions of the sugar moieties at positions 7, 9, 10, and 11 starting from the 5' end of the sense strand is fluorine. For example, the 2' positions of the sugar moieties at positions 7, 9, 10, and 11 starting from the 5' end of the sense strand are all fluorine.

In some embodiments, except for the 7th, 9th, 10th, and 11th nucleotides starting from the 5' end of the sense strand, at least one of the 2' positions of the remaining nucleotide sugar groups is a methoxy group.

In some embodiments, at least one of the 2' positions of the nucleotide sugars at positions 3, 5, 7, 9, 10, 11, 13, and 15 starting from the 5' end of the sense strand is fluorine. For example, the 2' positions of the nucleotide sugars at positions 3, 5, 7, 9, 10, 11, 13, and 15 starting from the 5' end of the sense strand are all fluorine.

In some embodiments, except for the 3rd, 5th, 7th, 9th, 10th, 11th, 13th, and 15th nucleotides starting from the 5' end of the sense strand, at least one of the 2' positions of the remaining nucleotide sugar groups is a methoxy group.

In some embodiments, except for the 7th, 8th, 9th, and 10th nucleotides starting from the 5' end of the sense strand, at least one of the 2' positions of the remaining nucleotide sugar groups is a methoxy group.

For example, the -OH at the 2' position of some or all of the nucleotide sugar groups of the sense strand and/or antisense strand can be substituted, wherein the substituent group is fluorine or methoxy, preferably the 2' position of the nucleotide sugar groups at positions 9, 10, and 11 from the 5' end of the sense strand is fluorine, and the 2' position of the nucleotide sugar groups at positions 2, 4, 6, 12, 14, 16, 18, and 20 from the 5' end of the antisense strand is fluorine, and the 2' positions of the remaining nucleotide sugar groups are all methoxy, or preferably the 2' position of the nucleotides at positions 5, 7, 8, and 9 from the 5' end of the sense strand is fluorine, and the 2' position of the nucleotide sugar groups at positions 2, 4, 8, 14, and 16 from the 5' end of the antisense strand is fluorine, and the 2' positions of the remaining nucleotide sugar groups are all methoxy.

In some embodiments, there are at least two consecutive phosphorothioate bonds between nucleotides of the sense strand and/or antisense strand.

In some embodiments, at least two consecutive phosphorothioate bonds exist between three consecutive nucleotides at at least one end of the sense strand end and/or the antisense strand end.

For example, there are at least two consecutive phosphorothioate bonds between three consecutive nucleotides at the 5' end and 3' end of the sense strand and the antisense strand.

For another example, the 2' position of the nucleotide sugar group at positions 9, 10, and 11 from the 5' end of the sense strand is fluorine, and the 2' position of the nucleotide sugar group at positions 2, 4, 6, 12, 14, 16, 18, and 20 from the 5' end of the antisense strand is fluorine, and the 2' position of the remaining nucleotide sugar groups are all methoxy, and there are at least two consecutive phosphorothioate bonds between three consecutive nucleotides at the 5' and 3' ends of the sense and antisense strands.

In some embodiments, the 2' position of some nucleotides of the sense strand is fluorine or methoxy, and at least three phosphate bonds between adjacent nucleotides at the end of the antisense strand can be thiolated. The 2' position of the 5th, 7th, 8th, 9th or the 3rd, 5th, 7th, 8th, 9th, 11th, 13th, 15th nucleotides starting from the 5' end of the sense strand is fluorine, and the 2' position of the remaining nucleotides is methoxy, and at least three phosphate bonds between adjacent nucleotides at the end of the antisense strand can be thiolated.

In some embodiments, the 2' position of some nucleotides of the sense strand is fluorine or methoxy, and at least three phosphate bonds between adjacent nucleotides at the end of the antisense strand can be thiolated. The 2' position of the 9th, 10th, 11th or the 3rd, 5th, 7th, 8th, 9th, 11th, 13th, 15th and/or 17th nucleotides starting from the 5' end of the sense strand is fluorine, and the 2' position of the remaining nucleotides is methoxy, and at least three phosphate bonds between adjacent nucleotides at the end of the antisense strand can be thiolated.

In some embodiments, the RNA inhibitor of the CFB modified sequence of the present invention is selected from the following Table 2-1, the RNA inhibitor of the C5 modified sequence is selected from the following Table 2-2, and the RNA inhibitor of the C3 modified sequence is selected from the following Table 2-3:

Table 2-1 RNA inhibitors with CFB modified sequences

Table 2-2 RNA inhibitors with C5 modified sequences

Table 2-3 RNA inhibitors with C3 modified sequences

Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Ts = 2'-O-methyl-3'-thiothymidylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-O-methyl-3'-thioguanylate =2'-fluorouridine, fC =2'-fluorocytidine, fT =2'-fluorothymidine; fGs =2'-fluoro-3'-thioguanylate, fAs =2'-fluoro-3'-thioadenylate, fUs =2'-fluoro-3'-thiouridylate, fCs =2'-fluoro-3'-thiocytidine, T = deoxythymidine, dA =2'-deoxy-adenylate, invAb is inverted deoxy abasic cap, Tgn is glycol deoxythymidine.

In some embodiments, the sense strand or antisense strand of the RNA inhibitor described in the present invention has a sequence of at least 15 consecutive nucleotides identical to the sense strand or antisense strand in Tables 1 to 2, or a sequence that differs by one, two or three nucleotides.

In some embodiments, the distribution, targeting or stability of the RNA inhibitor is altered by introducing a ligand for a target tissue receptor into the vector. For example, a specific ligand can provide enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cell or organ compartment, a body tissue, an organ or region)) compared to a species in which the ligand is not present.

The ligand can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example a synthetic polyamino acid.

The ligand can also include a targeting group, such as a cell or tissue targeting agent that is combined with a specified cell type such as a kidney cell, such as a lectin, a glycoprotein, a lipid or a protein, such as an antibody. The targeting group can be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, a surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, polyglutamic acid, polyaspartic acid, a lipid, cholesterol, a steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In some embodiments, the ligand is a multivalent galactose, such as N-acetyl-galactosamine.

The sense strand and antisense strand contained in the RNA inhibitor of the present invention can be conveniently and routinely prepared by the well-known technique of solid phase synthesis. Any other method known in the art for such synthesis, such as liquid phase synthesis or fermentation, can be used additionally or alternatively.

In some embodiments, in addition to commercially available and conventionally used standard nucleoside phosphoramidite monomers and non-standard nucleoside phosphoramidite monomers, the sense strand and antisense strand contained in the RNA inhibitor of the present application can be synthesized by an automatic synthesizer using a phosphoramidite method derived from a carrier-nucleoside phosphoramidite monomer.

In some embodiments, the ligand described in the present invention is coupled to the 5' end and/or 3' end of the antisense chain, and/or the 5' end and/or 3' end of the sense chain through a carrier structure.

For example, the carrier structure can be coupled to the 5' end and/or the 3' end of the sense strand; or the carrier structure can be coupled to the 5' end of the antisense strand and the carrier structure can be coupled to the 3' end of the sense strand; or the carrier structure can be coupled to the 3' end of the antisense strand and the ligand can be coupled to the 5' end of the sense strand.

In some embodiments, the carrier structure includes 5'MVIP and 3'MVIP, wherein the 5'MVIP is coupled to the 5' end of the sense strand and/or the antisense strand, and the 3'MVIP is coupled to the 3' end of the antisense strand and/or the sense strand, the structure of the 5'MVIP is as shown in Formula I, and the structure of the 3'MVIP is as shown in Formula II.

(XL) _n -BDR ₁ -,

I

(XL) _m - _BDR2- ,

II

in,

X is a liver-targeting specific ligand;

L is a branched chain;

B is a connector;

D is the connecting chain;

R ₁ and R ₂ are transfer points;

The 5'MVIP is connected to the 5' end of the sense strand or the 5' end of the antisense strand through the transfer point _R1 , and the 3'MVIP is connected to the ₃ ' end of the sense strand or the 3' end of the antisense strand through the transfer point R2, n and m are each independently any integer from 0 to 4, and n+m=an integer from 2 to 6, preferably n+m=2, 3 or 4, and more preferably 4.

In some embodiments, the connection between R ₁ or R ₂ and the sense strand or antisense strand is through phosphate or modified phosphate, and R ₁ or R ₂ is preferably connected to the sense strand or antisense strand through phosphate or phosphorothioate.

In some embodiments, m or n may be 0, i.e., there is no 3'MVIP or 5'MVIP.

In some embodiments, when n=0 (ie, there is no 5'MVIP), the structure of the 3'MVIP may be:

In some embodiments, when n=1, the structure of the 3'MVIP may be:

In some embodiments, when n=2, the structure of the 3'MVIP may be:

In some embodiments, when n=3, the structure of the 3'MVIP may be:

In some embodiments, when n=4, the structure of the 3'MVIP may be:

In some embodiments, the n refers to the sum of the n placed in the 5'MVIP at the 5' end of the sense strand and the antisense strand of the RNA inhibitor, and the m refers to the sum of the m placed in the 3'MVIP at the 3' end of the sense strand and the antisense strand of the RNA inhibitor.

In some embodiments, the _R1 and _R2 structures contain -NH-, -S- and/or -O-, and _R1 and _R2 are connected to the connecting chain D and the 5' end and 3' end of the sense chain and/or antisense chain respectively through the -NH-, -S- or -O- in the structure, and _R1 and _R2 are the same or different.

In some embodiments, _R1 and _R2 are optionally straight carbon chains, or straight carbon chains with amide, carboxyl or alkyl branches, or cyclic structures, wherein the cyclic structure includes a saturated or unsaturated aliphatic carbocyclic group, or a five-membered or six-membered heterocyclic group or aromatic hydrocarbon group containing sulfur, oxygen or nitrogen atoms.

In some embodiments, R ₁ and/or R ₂ is -E ₁ (CH ₂ ) _x CH ₂ E ₂ -, wherein x is any integer from 3 to 12, and the groups E ₁ and E ₂ can be -NH-, -S- or -O-, respectively.

In some embodiments, _R1 and/or _R2 is _-E1 ( _CH2 ) _x1CH (OH)( _CH2 ) _x2E2- , wherein x1 or x2 is each independently any integer from 3 to 10, _{and E1} and _E2 can be -NH-, -S- or -O-, respectively.

In some embodiments, the R ₁ is a heterocyclic or carbocyclic structure containing N, S or O as shown below:

In some embodiments, the transition point _R1 is -NH( _CH2 ) _xCH2O- , wherein x is any integer from 3 to 12, preferably any integer from 4 to 6, and can _be introduced by the following two phosphoramidite monomers.

One -O- or -S- in the iR ₁ structure is used for the synthesis of the R ₁ phosphoramidite monomer, which is connected to the 5' end of the sense chain or antisense chain of the RNA inhibitor by solid phase synthesis. The -NH-, -S- or -O- in the structure is used to connect with the connecting chain D in the 5'MVIP, thereby introducing the liver-targeting specific ligand X at the 5' end of the sense chain or antisense chain in the RNA inhibitor. The exemplary structure of the monomer introduced into the 5' end of the sense chain or antisense chain of the RNA inhibitor is as follows:

In some embodiments, the following structures are preferred:

ii. One -NH-, -S- or -O- in the _R1 structure is first connected to the connecting chain D, and the other -NH-, -S- or -O- is used to form an ester with the phosphoramidite in the synthesis of the 5'MVIP phosphoramidite monomer. The structure of the sense chain or antisense chain 5'MVIP phosphoramidite monomer is exemplified as follows:

In some embodiments, the 5'MVIP phosphoramidite monomer of the sense strand or antisense strand preferably has the following structure:

When n in the general formula is 1-4, the linker B part in the monomer is branched 1 to 4 times to obtain the corresponding monomer compound. With the help of the monomer compound, the liver-targeting specific ligand X is introduced into the 5' end of the sense chain or the antisense chain through solid phase synthesis.

In some embodiments, the transition point _R1 is -NH( _CH2 ) _xCH2O- , wherein x can be any integer from 3 to ₁₂ , preferably any integer from 4 to 6.

In some embodiments, the 5'MVIP phosphoramidite monomer structure is selected from the following structures:

In some embodiments, the transition point _R2 is a heterocyclic or carbocyclic structure containing N, S or O as shown below:

In some embodiments, the transition point _R2 is -NH( _CH2 ) _x1CH (OH)( _CH2 ) _x2CH2O- , wherein x1 is any integer from 1 to 4, _and x2 is any integer from 0 to 4.

The transfer point _R2 described in the present application is formed by esterification or amide formation between succinic anhydride and -NH-, -S- or -O- in the _R2 structure, and at the same time, coupling with -NH- in the blank Solid Support to form a 3'MVIP solid spport, and then introducing 3'MVIP into the 3' end of the sense chain or antisense chain through the phosphoramidite solid phase synthesis method.

In some embodiments, the heterocyclic ring in the transition point _R2 structure is a pyrrole ring or a piperidine ring, which is connected to the connecting chain D of 3'MVIP through the nitrogen heteroatom in the ring. The exemplary structure of the introduced 3'MVIP solid spport is as follows:

When m in the general formula is 1-4, the connector B part in the monomer is branched 1 to 4 times to obtain the corresponding Solid Support.

In some embodiments, the transition point _R2 is _-B4 ( _CH2 ) _x1CH (OH)( _CH2 ) _x2CH2B5- , wherein x1 is any integer from ₁ to ₄ , x2 is any integer from 0 to 4, _B4 and _B5 are -NH-, -S- or -O-, respectively, and the exemplary structure of the introduced 3'MVIP solid spport is as follows:

When m in the general formula is 1-4, the connector B part in the monomer is branched 1 to 4 times to obtain the corresponding Solid Support.

In some embodiments, R ₂ is -NHCH ₂ CH(OH)CH ₂ O-, and the exemplary structure of the introduced 3'MVIP solid spport is as follows:

When m in the general formula is 1-4, the connector B part in the monomer is branched 1 to 4 times to obtain the corresponding Solid Support.

In some embodiments, the 3'MVIP solid support structure is as follows:

In some embodiments, the liver-targeting specific ligand X is selected from structures used to enhance the uptake of RNA inhibitors by hepatocytes, and may be lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimetic structures. In the RNA inhibitors provided in the present application, the liver-targeting specific ligands X introduced into the ends of the sense strand or antisense strand of the RNA inhibitor may be the same or different. For example, in terms of properties, some may be structures that enhance liver targeting, some may be structures that regulate the pharmacokinetics of the RNA inhibitor in vivo, and some may be structures that have in vivo dissolution activity. In some embodiments, the liver-targeting specific ligand X is selected from one or more monosaccharides and their derivatives in the following structures.

In some embodiments, the monosaccharide is selected from one or more of the following structures: mannose, galactose, D-arabinose, glucose, fructose, xylose, glucosamine, ribose. The monosaccharide derivative is selected from mannose derivatives, galactose derivatives, glucose derivatives, ribose derivatives and other derivatives.

In some embodiments, the liver-targeting specific ligand X is selected from galactose, galactosamine, N-acetylgalactosamine and derivatives thereof, and its general structural formula is as follows:

Wherein, _W1 is hydrogen or a hydroxyl protecting group, which may be the same or different; W is -OH, -NHCOOH or -NHCO( _CH2 ) _{qCH3 ,} wherein q is an integer of 0-4; _W2 is -NH-, O, S or C.

In some embodiments, the liver-targeting specific ligand X is N-acetylgalactosamine and its derivatives.

In some embodiments, the liver-targeting specific ligand X is selected from the following structures:

Wherein, W is selected from one or two of -OH, -NHCOOH or -NHCO(CH ₂ ) _q CH ₃ , wherein q is an integer of 0-4.

In some embodiments, the liver-targeting specific ligand X in the same 5'MVIP or 3'MVIP structure may be the same or different.

In some embodiments, X between 5'MVIP and 3'MVIP may be the same or different.

In some embodiments, the branched chain L is a C 4 -C 18 carbon chain containing -NH-, -C(=O)-, -O-, -S-, an amide group, a phosphoryl group, a thiophosphoryl group, a C ₄ -C _{10 aliphatic carbocyclic} group, a phenyl group, or a combination of these groups.

In some embodiments, the branched chain L further has a hydroxyethyl group or a carboxylic acid side chain.

In some embodiments, the branched chain L is a C ₇ -C ₁₈ carbon chain containing an amide group or a six-membered aliphatic carbocyclic group.

In some embodiments, the side chain L is selected from one or more of the following structures:

Wherein, r1 is any integer of 1-12, r2 is any integer of 0-20, and Z is H, an alkyl group or an amide group, and the alkyl group is, for example, a C ₁ -C ₅ alkyl group.

In some embodiments, the structure of the linker B is related to the number of Xs that can be introduced. The linker B contains -NH-, C, O, S, amide, phosphoryl, thiophosphoryl, and when n or m is 1, it is a straight carbon chain. When n or m is 2, 3 or 4, the number of forks is 2, 3 or 4, respectively.

In some embodiments, the linker B is selected from the following structures:

wherein _A1 and _A2 are each independently C, O, S, -NH-, carbonyl, amide, phosphoryl or thiophosphoryl, and r is an integer of 0-4.

In some embodiments, the linker B is selected from the following structures:

Wherein, r is any integer from 0 to 4.

In some embodiments, the linker B is selected from the following structures:

In some embodiments, the linker B is selected from the following structures:

In some embodiments, the connecting chain D is a C 3 -C 18 carbon chain containing -NH-, C=O, O, S, amide, phosphoryl, thiophosphoryl, aromatic hydrocarbon, C ₄ -C ₁₀ aliphatic carbocyclic group, five-membered or six-membered heterocyclic group containing _{1-3 nitrogen} atoms, or a combination of these groups.

In some embodiments, the connecting chain D further has a side chain of a hydroxymethyl group, a methyl tert-butyl group, a methylphenol group, or a C ₅ -C ₆ aliphatic ring group.

In some embodiments, the connecting chain D is a C ₃ -C ₁₀ carbon chain containing two C═O groups, a six-membered aliphatic carbocyclic group, or a phenyl group.

In some embodiments, the connecting chain D is a C ₃ -C ₁₀ carbon chain containing two C═O groups.

In some embodiments, the connecting chain D is selected from the following structures:

Wherein, each p is independently any integer of 1-20; s is an integer of 2-13; _Z1 and _Z2 are the same or different substituent groups, such as _C3 - _C10 alkyl.

In some embodiments, the connecting chain D is selected from the following structures:

In some embodiments, the connecting chain D is selected from the following structures:

In some embodiments, the (XL) _n -BD- in the 5'MVIP structure and the (XL) _m -BD- in the 3'MVIP structure are selected from one or more of the following structures:

In some embodiments, the X, L, B and D are the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP.

In some embodiments, the (XL) _n -BD- in the 5'MVIP structure is selected from the structures shown in Table 3:

Table 3 (XL) _n -BD-structure of 5'MVIP

In some embodiments, 5'MVIP may not exist, in which case m can be any integer from 2 to 4.

In some embodiments, the (XL) _m -BD- in the 3'MVIP structure is selected from the structures shown in Table 4:

Table 4 (XL) _m -BD-structure of 3'MVIP

In some embodiments, the combination of (XL) _n -BD- and R ₁ in the carrier structure 5′MVIP is as shown in Table 5.

Table 5 Combinations of (XL) _n -BD- and R ₁ in 5'MVIP

In some embodiments, 3'MVIP may not exist, in which case n may be any integer from 2 to 4.

In some embodiments, the combination of (XL) _m -BD- and R ₂ in the carrier structure 3'MVIP is as shown in Table 6.

Table 6 Combinations of (XL) _m -BD- and R ₂ of 3'MVIP

In some embodiments, the 5’MVIP is selected from any one or more of 5’MVIP01 to 5’MVIP22 in Table 5.

In some embodiments, the 3’MVIP is selected from any one or more of 3’MVIP01 to 3’MVIP27 in Table 6.

In some embodiments, there is a possibility of combining a 5'MVIP in Table 5 with any of the 3'MVIPs in Table 6, where n+m=2, 3, 4, 5 or 6.

In some embodiments, the sense strand and antisense strand coupled to the carrier in the CFB RNA inhibitor can be selected from the following Table 7-1, the sense strand and antisense strand coupled to the carrier in the C5 RNA inhibitor can be selected from the following Table 7-2, and the sense strand and antisense strand coupled to the carrier in the C3 RNA inhibitor can be selected from the following Table 7-3:

Table 7-1 Sense strand or antisense strand coupled to carrier in CFB RNA inhibitor

Table 7-2 Sense strand or antisense strand coupled to carrier in C5 RNA inhibitor

Table 7-3 Sense strand or antisense strand coupled to carrier in C3 RNA inhibitor

In some embodiments, the sense strand and antisense strand of the RNA inhibitor described in the present application have a sequence of at least 15 consecutive nucleotides that are the same as the sense strand and antisense strand in Table 7, or a sequence that differs from the sense strand and antisense strand in Table 7 by one, two or three nucleotides.

It should be emphasized that the combination of the sense chain and the antisense chain is not limited to the double-strand combination in Table 7. One of the sense chains in Table 7 can be complementary to any antisense chain; for example, GsUsCUAGfUCfAfAfCUUAAUUGAsGsA (SEQ ID NO: 554) in Table 7-1 can be complementary to UsfCsUfCAfAUfUAAGUUfGAfCUAGACsAsC (SEQ ID NO: 583), UsfCsUfCAfAUfUAAGUUfGAfCUAGsAsC (SEQ ID NO: 584), UsdCsUCdAATUAAGTUfGACUAGA CsAsC (SEQ ID NO: 588), UsfCsUCAfAUfUfAAGUUfGAfCUAGACsAsC (SEQ ID NO: 591), UsfCsUfCAfAUfUAAGUUfGAfCUAGACsAsCTT (SEQ ID NO: 593), and UsfCsUCAfAUfUfAAGUUfGAfCUAGACsAsC (SEQ ID NO: 594) are complementary pairs. Any sense chain or antisense chain in the present invention is an independent individual. The combination method is not limited. At least 85% of the nucleotides can be complementary paired and can form a double-stranded inhibitor.

In some embodiments, any sense strand or antisense strand of the present invention can be connected to carriers of different structures. As an example, the 5' end and/or 3' end of any sense strand or antisense strand of the present invention are connected to 5'MVIP and/or 3'MVIP of different structures, and the coupling combination is not limited.

In some embodiments, the double-stranded RNA inhibitor of the present invention can be optionally conjugated with one or more ligands, and the ligands can be applied to the double-stranded RNA inhibitor of the present invention as long as they can enhance the activity, cellular distribution or cellular uptake (e.g., entering cells) of the double-stranded RNA inhibitor. The ligand can be connected to the sense strand, antisense strand or both strands at the 3' end, 5' end or both ends. The carrier is not limited to the MVIP listed in the present invention, and may also include but is not limited to: GalNac carriers of any structure, cationic lipid carriers, viral vectors, lipophilic parts, amphiphilic parts, targeting groups, small molecule drugs, proteins, peptides, antibodies.

In some embodiments, the antisense strand of the RNA inhibitor of the present invention can be obtained by coupling the antisense strands in Tables 1 and 2 with 5'MVIP and/or 3'MVIP.

In some embodiments, the antisense strand of the RNA inhibitor described in the present invention has a sequence of at least 15 consecutive nucleotides identical to the antisense strand in Tables 1-2, or a sequence that differs from the antisense strand in Tables 1-2 by one, two or three nucleotides coupled to 5'MVIP and/or 3'MVIP.

In some embodiments, the double-stranded RNA inhibitor of the present invention can be optionally conjugated with one or more ligands, and the ligands can be applied to the double-stranded RNA inhibitor of the present invention as long as they can enhance the activity, cellular distribution or cellular uptake (e.g., entering cells) of the double-stranded RNA inhibitor. The ligand can be connected to the sense strand, antisense strand or both strands at the 3' end, 5' end or both ends. The carrier is not limited to the MVIP listed in the present invention, and may also include but is not limited to: GalNac carriers of any structure, cationic lipid carriers, viral vectors, lipophilic parts, amphiphilic parts, targeting groups, small molecule drugs, proteins, peptides, antibodies.

Patent CN113171371B examines in detail the effects of different X, L, B, D, _R1 and _R2 in the 5'MVIP and/or 3'MVIP structures on the activity of RNA inhibitors, and the entire text of the patent is incorporated into the present invention.

When X is galactose, galactosamine, N-acetylgalactosamine and its derivatives, among the RNA inhibitors provided by the present invention, N-acetylgalactosamine and its derivatives are preferably used as liver-targeting specific ligands, as shown in Table 8:

Table 8

The length of L has a great influence on the effect of RNA inhibitors. The L chain should be neither too short nor too long. When containing -NH-, C=O, O, S, amide, phosphoryl, thiophosphoryl, aliphatic carbocyclic group such as cyclohexane or a combination of these groups, or in the same 5'MVIP, 3'MVIP structure or when 5'MVIP and 3'MVIP have different L structures, the activity of the obtained RNA inhibitors is not much different within the range of carbon chain length of C7-C18, as shown in Table 9.

Table 9

Except for the change in the structure of linker B, when X, L, D and _R1 / _R2 are consistent with those in the combination 5'MVIP09/3'MVIP09, _A1 and _A2 in the general formula of linker B are each independently C, O, S, -NH-, carbonyl, amide, phosphoryl or thiophosphoryl, r is any integer from 0 to 4, and when linker B is the same or different between 5'MVIP and 3'MVIP, the resulting RNA inhibitor activity is not much different.

Table 10

When the MVIP structure and RNA inhibitor are the same, different connecting chains D will affect the activity of the RNA inhibitor, among which the effects of D1, D2, and D4 are close to and better than D3. The structure of D is shown in Table 11.

Table 11

Different transfer points _R1 will affect the activity of RNA inhibitors, among which R1-1 as the transfer point has the best RNA inhibitor activity. The structure of _R1 is shown in Table 12.

Table 12

Different transfer points _R2 will affect the activity of RNA inhibitors, among which R2-1 is the best RNA inhibitor when used as the transfer point. The _R1 structure is shown in Table 13.

Table 13

In some embodiments, n+m in the RNA inhibitor described in the present invention is 2, 3, 4, 5 and 6 respectively. The positions for coupling of 5'MVIP and/or 3'MVIP include the 5' end and/or 3' end of the antisense chain, the 5' end and/or 3' end of the sense chain, the 5' end of the antisense chain and the 3' end of the sense chain, and the 5' end of the sense chain and the 3' end of the antisense chain.

In some embodiments, n+m in the RNA inhibitor of the present invention is 2, 3, 4, 5 and 6 respectively. The positions of 5'MVIP and/or 3'MVIP coupling include the 5' end and/or 3' end of the antisense strand in Table 1-Table 2, the 5' end and/or 3' end of the sense strand in Table 1-Table 2, the 5' end of the antisense strand and the 3' end of the sense strand in Table 1-Table 2, and the 5' end of the sense strand and the 3' end of the antisense strand in Table 1-Table 2. The obtained 5'MVIP and 3'MVIP combinations are shown in Table 14:

Table 14 5'MVIP and 3'MVIP combination list

In some embodiments, n and m are each independently any integer of 0-4, preferably each independently an integer of 1-3, and n+m=2-6, preferably n+m=2, 3 or 4, more preferably 4.

In some embodiments, the RNA inhibitor described herein or a pharmaceutically acceptable salt thereof is preferably prepared or synthesized in the form of a sodium salt, a triethylamine salt or other pharmaceutically acceptable salts.

In some embodiments, the RNA inhibitor described herein or a pharmaceutically acceptable salt thereof is more preferably a sodium salt or a triethylamine salt thereof.

On the other hand, the present application also provides a pharmaceutical composition comprising the RNA inhibitor or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides a pharmaceutical composition comprising the RNA inhibitor or a pharmaceutically acceptable salt thereof and an optional pharmaceutically acceptable excipient. The RNA inhibitor provided by the present invention or a pharmaceutically acceptable salt thereof is used in the preparation of a medicament for treating and/or preventing a disease associated with an elevated level of the complement system, including but not limited to lipid metabolism disorders.

In some embodiments, the complement system regulates the level of triglycerides in plasma by inhibiting the activity of LPL (hepatic lipase) in the liver and adipose tissue, limiting the release of fatty acids. It affects insulin resistance, fat metabolism and overall energy balance. Factors that may inhibit the expression of complement system mRNA in vivo and/or in vitro include PPARδ, statins, insulin, leptin, thyroid hormones and lipopolysaccharides, etc. 5-10; the inhibitors of the present invention can be used in combination with these drugs, and there is a prospect of further improving the therapeutic effect of lipid metabolism, sugar metabolism, cardiovascular disease, etc.

One example approach is to formulate the composition for systemic administration by parenteral delivery, for example, subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery.The pharmaceutical compositions provided herein can be administered at a dose sufficient to inhibit complement system gene expression.

A pharmaceutically acceptable "excipient" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. Excipients can be liquid or solid and are selected taking into account the planned mode of administration to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components in a given pharmaceutical composition. The RNA inhibitors described herein can be delivered in a manner that targets specific tissues (e.g., hepatocytes).

In some embodiments, the pharmaceutical composition of the present invention further comprises a delivery vehicle (such as nanoparticles, dendrimers, polymers, liposomes or cationic delivery systems).

In some embodiments, the delivery vehicle described herein comprises a liposome.

In some embodiments, the delivery vehicle described herein includes nanolipids that can form liposome-nucleic acid nanoparticles with nucleic acid molecules.

In some embodiments, the delivery vehicle described herein comprises the amphiphilic lipid compound M10C1.

The pharmaceutical compositions provided by the present invention include, but are not limited to, solutions, emulsions, and formulations containing liposomes. These compositions can be produced from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. The formulations include those targeted to the liver. The pharmaceutical formulations of the present application, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques known to the pharmaceutical industry. Such techniques include the step of combining the active ingredient with a pharmaceutically acceptable adjuvant or excipient.

use

On the other hand, the present application provides a method for reducing the expression of complement system mRNA or protein in cells or tissues, which comprises contacting the cells or tissues with an effective amount of the aforementioned RNA inhibitor that inhibits complement system gene expression or a pharmaceutically acceptable salt thereof, and/or the aforementioned pharmaceutical composition.

Cells suitable for treatment using the methods of the present application may be any cells expressing complement system genes, for example, liver cells, brain cells, gallbladder cells, heart cells or kidney cells, but preferably liver cells. Cells suitable for use in the methods of the present application may be mammalian cells, and when contacted with cells expressing complement system genes, the RNA inhibitor inhibits the expression of complement system genes (e.g., human, primate, non-primate or rat complement system genes) by at least about 50%, for example, as determined by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis, Western blotting or flow cytometry.

In some embodiments, the tissue is liver tissue.

In some embodiments, the cells and tissues are ex vivo.

In some embodiments, the cells and tissues are in a subject.

As used herein, the term "inhibit" can be used interchangeably with "reduce," "lower," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition. The expression of complement system genes can be evaluated based on the level or level change of any variable related to the expression of complement system genes, for example, the level of complement system mRNA. This level can be analyzed in a single cell or in a cell population (including, for example, a sample derived from a subject). The control level can be any type of control level used in the art, for example, a baseline level before administration or a level measured from a similar subject, cell or sample that has not been treated or treated with a control (such as, for example, a buffer-only control or an inactive agent control).

Inhibition of complement system gene expression can be manifested by a decrease in the amount of mRNA expressed by a first cell or cell population (such cells may, for example, be present in a sample derived from a subject) in which complement system genes are transcribed and have been treated (e.g., by contacting one or more cells with an RNA inhibitor of the present application, or by administering an RNA inhibitor of the present application to a subject in which the cells are present) such that expression of the complement system genes is inhibited, as compared to a second cell or cell population that is substantially identical to the first cell or cell population but has not been so treated (control cells that have not been treated with an RNA inhibitor or have not been treated with an RNA inhibitor targeting a gene of interest).

In a preferred embodiment, the evaluation is performed by using appropriate concentrations of siRNA in cell lines that highly express the complement system, and the mRNA levels in the treated cells are expressed as a percentage of the mRNA levels in non-treated control cells.

In other embodiments, inhibition of complement system gene expression can be assessed by a reduction in a parameter functionally associated with complement system gene expression, e.g., complement system levels in the blood or serum of a subject. Complement system gene inhibition can be measured in any cell expressing the complement system (endogenous or exogenous from an expression construct) and by any assay known in the art.

Inhibition of complement system expression can be manifested by a decrease in the level of complement system expressed by a cell or population of cells or a sample from a subject (eg, protein levels in a blood sample derived from a subject).

Control cells, cell groups or subject samples that can be used to evaluate complement system gene inhibition include cells, cell groups or subject samples that have not been contacted with the RNA inhibitor of the present application. For example, control cells, cell groups or subject samples can be derived from a single subject (e.g., a human or animal subject) or an appropriately matched population control before treatment with an RNA inhibitor.

The level of complement system mRNA expressed by a cell or cell population can be determined by any method known in the art for evaluating mRNA expression. For example, qRT-PCR is used to evaluate the reduction of gene expression. The reduction of protein production can be evaluated by any method known in the art, for example, ELISA. In some embodiments, a puncture liver biopsy sample is used as a tissue material for monitoring the reduction of complement system gene expression. In other embodiments, a blood sample is used as a subject sample for monitoring the reduction of complement system expression.

On the other hand, the present application provides the use of the aforementioned RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition in the preparation of a drug for preventing and/or treating a disease or condition or reducing the risk of a disease or condition.

On the other hand, the present application provides a method for preventing and/or treating a disease or condition, comprising administering to a subject in need thereof an effective amount of the aforementioned RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, and/or the aforementioned pharmaceutical composition.

The in vivo method of the present application may include administering to a subject a pharmaceutical composition comprising an RNA inhibitor, wherein the RNA inhibitor comprises a nucleotide sequence complementary to at least a portion of a complement system mRNA of a mammal to which the RNA inhibitor is administered. The pharmaceutical composition of the present invention may be administered in any manner known in the art, including, but not limited to: oral, intraperitoneal or parenteral routes, including intracranial (e.g., intraventricular, intracerebral parenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical composition is administered by intravenous infusion or injection. In some embodiments, the pharmaceutical composition is administered by subcutaneous injection. In some embodiments, the composition is administered by intramuscular injection.

The RNA inhibitors provided herein can also be administered as "free RNA inhibitors". Free RNA inhibitors are administered in the absence of a pharmaceutical composition. Naked RNA inhibitors can be in a suitable buffer. The buffer can contain acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer containing the RNA inhibitor can be adjusted so as to be suitable for administration to a subject.

Alternatively, the RNA inhibitors provided herein can be administered as a pharmaceutical composition, such as a liposomal formulation.

The pharmaceutical composition provided herein can be administered at a dosage sufficient to inhibit complement system gene expression. Typically, the suitable dosage of the RNA inhibitor described herein is in the range of about 0.001 to about 200.0 mg per kilogram of subject body weight per day, typically in the range of about 1 to 50 mg per kilogram of body weight per day. Typically, the suitable dosage of the RNA inhibitor described herein is in the range of about 0.1 mg/kg to about 5.0 mg/kg, for example, in the range of about 0.3 mg/kg to about 3.0 mg/kg.

In some embodiments, the method further comprises determining the level of the complement system in a sample from the subject.

For example, the method further comprises determining the level of the complement system in a blood sample, a serum sample, or a urine sample from the subject.

In yet another aspect, the present application provides a cell comprising the aforementioned RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof.

On the other hand, the present application provides a drug kit comprising the aforementioned RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the RNA inhibitors, preparation methods and uses provided by the present application, and are not intended to limit the scope of the present invention.

Example

illustrate:

The Chinese name of DMSO is dimethyl sulfoxide;

The Chinese name of DMF is N,N-dimethylformamide;

The Chinese name of HOBt is 1-hydroxybenzotriazole;

The Chinese name of HBTU is O-benzotriazole-tetramethyluronium hexafluorophosphate;

The Chinese name of DIPEA (DIEA) is N,N-diisopropylethylamine;

The Chinese name of DCM is dichloromethane;

The Chinese name of DMAP is 4-dimethylaminopyridine;

The Chinese name of DMT-CL is 4,4'-dimethoxytriphenylmethane;

The Chinese name of MEOH is methanol;

The Chinese name of TBTU is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate;

The name of the solid phase carrier, such as macroporous aminomethyl resin (Resin).

Example 1 Synthesis of RNA Inhibitors

The sense strand and antisense strand of the uncoupled carrier structure are synthesized by a standard solid phase phosphoramidite method using a multi-channel solid phase synthesizer, and then the sense strand is complementary annealed with the corresponding antisense strand to prepare the corresponding RNA inhibitor.

The basic steps of the solid phase phosphoramidite method include:

1) Deprotection: remove the Solid Support hydroxyl protecting group (DMTr) in the starting monomer;

2) Coupling: Add the first phosphoramidite monomer and the coupling reaction occurs from 3' to 5' direction;

3) Oxidation: oxidizing the obtained nucleoside phosphite to a more stable nucleoside phosphate (i.e., oxidizing trivalent phosphorus to pentavalent phosphorus);

4) Blocking: Cap the 5’-OH of the failed nucleotide sequence in the previous step to prevent it from further participating in the reaction; repeat the above steps until the last phosphoramidite monomer is connected; then use methylamine aqueous solution and ammonia water to cleave the ester bond between Solid Support and the starting monomer, and remove the protecting groups on each base and phosphate on the resulting nucleotide sequence; separate and purify by HPLC, filter and sterilize, and lyophilize to obtain the corresponding sense chain or antisense chain.

Description of the synthesis process of RNA inhibitors:

Dissolve the sense strand and antisense strand freeze-dried powders separately, mix them in equal moles, add an appropriate amount of water for injection, and add an appropriate amount of TRIS buffer solution. Shake gently for about 1 to 2 minutes to mix the solution evenly. Heat the water bath to 92°C to 95°C. Heat the reaction solution in a water bath for 3 to 5 minutes, and shake gently to heat the solution evenly. Cool naturally to room temperature. Obtain a colorless or slightly yellow transparent liquid, take a sample for inspection, and measure the concentration.

Example 2-1 In vitro study on the inhibition of complement system gene expression by CFB and C5 RNA inhibitors

The RNA inhibitors of this example are selected from Table 1-1 and Table 1-2, and are prepared by the method described in Example 1.

Hep3B and huh7 cells were digested with trypsin and adjusted to the appropriate density, then seeded into 96-well plates at a viable cell concentration of 0.2×10 ⁶ cells/mL. At the same time as seeding, cells were transfected with test siRNA or control siRNA using Lipofectamine RNAiMax (Invitrogen-13778150). siRNA was tested in triplicate at the following concentrations: 0.1nM, 0.02nM, 0.015nM, 0.001nM, 10nM, 1nM, 0.01nM, and a no siRNA control group and a positive control group containing only cells and RNAiMax were set up.

24 hours after transfection, the culture medium was removed and the cells were harvested for RNA extraction. Total RNA was extracted using a 96-channel fully automatic nucleic acid extraction and purification instrument or a Promega total RNA extraction kit.

According to the manual, use cDNA synthesis was performed using the II All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit. The target cDNA was detected by qPCR, and GAPDH cDNA was detected in parallel as an internal control. Fluorescence quantitative PCR was performed using a Thermo QuantStudio 1 instrument as follows: 30 seconds at 95°C, followed by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C.

Data Analysis:

The mRNA expression level of the target gene in the sample was calculated based on the Ct value of each sample using the ΔΔCt relative quantification method, and the relative expression of the target gene was expressed as 2 ^{- ΔΔCt} .

The equation is listed below:

ΔCT = target gene average Ct - GAPDH average Ct

ΔΔCT = ΔCT (sample) - ΔCT (random control or Lipofectamine RNAiMax control);

Relative quantification of target gene mRNA = 2 ^(-ΔΔCT)

Inhibition % = (relative quantification of control - relative quantification of sample) / relative quantification of control x 100%.

The results of the CFB RNA inhibitor transfection experiment in Hep3B cells are shown in Tables 15-1 to 15-4 below.

The results of the Hep3B cell transfection experiment with C5 RNA inhibitor are shown in Tables 15-5 to 15-11 below.

Table 15-1 Inhibitory effect of 0.1nM CFB RNA inhibitor on Hep3B cells

Table 15-2 Inhibitory effect of 0.02nM CFB RNA inhibitor on Hep3B cells

Table 15-3 Inhibitory effect of 0.015nM CFB RNA inhibitor on Hep3B cells

Table 15-4 Inhibitory effect of 0.1CFB RNA inhibitor on Hep3B cells

Table 15-5 Inhibitory effect of 1nM C5 RNA inhibitor on Huh7 cells

Table 15-6 1. Inhibitory effect of 0.1 nM C5RNA inhibitor on huh7 cells

Table 15-7 Inhibitory effect of 0.01nM C5 RNA inhibitor on Hep3B cells

Table 15-8 Inhibitory effect of 0.01nM C5 RNA inhibitor on Hep3B cells

Example 2-2 In vitro RNA inhibitor inhibition of C3 gene expression test

The RNA inhibitors of this example are shown in Tables 1-3. Plasmid DNA (C3_PSICHECK(TM)-2 plasmid) was transferred into Hep3B and Huh7 cells using Fugene HD. The transfected cells were inoculated into a 96-well plate at a density of 10,000 cells per well, and the culture medium in each well was 100 μL. The cells were placed in a 5% CO ₂ , 37°C incubator and cultured overnight. Then the RNA inhibitor was prepared with PBS to prepare the RNA inhibitor sample solution of nanolipids of the corresponding concentration. RNAiMAX/Opti-MEM was added to each well at the corresponding position, and the RNA inhibitor sample solution diluted with the corresponding concentration was added to the well, mixed and incubated, and the incubated mixture was mixed evenly with DMEM containing 10% FBS. The culture medium in each well was aspirated, and then a new culture medium containing the sample was added. After adding, it was placed in a 5% CO ₂ , 37°C incubator for culture, and the final concentration of the sample test was 0.01nM.

Take the cells out of the incubator, discard the supernatant, add fresh culture medium and detection reagent, shake in the dark, and after the cells are fully lysed, transfer the samples to a light-proof white plate to detect the luminescent signal of fireflies; add & Detection reagent, shake in the dark, detect Renilla luciferase signal. Calculate the ratio of the main reporter gene to the internal reference reporter gene signal in each well. The obtained test results are shown in Table 15-9 below.

Table 15-9 Inhibitory effect of 0.01 nM RNA inhibitor on C3 mRNA in Hep3B cells

Based on the screening results of the primary sequence, parts are selected for modification and carriers are added to further screen inhibitors.

Example 3 Synthesis of carrier structure

When the 3' end of the sense strand or antisense strand of the RNA inhibitor of the present application is coupled with the carrier structure 3'MVIP, the solid support of 3'MVIP is used as the starting monomer of solid phase synthesis. When the 5' end of the sense strand or antisense strand of the RNA inhibitor of the present application is coupled with the carrier structure 5'MVIP, the 5'MVIP phosphoramidite monomer is used as the last monomer of solid phase synthesis.

The solid spport formula of 3'MVIP is as follows:

When m is 1-4, the connector B part in the general formula is branched 1 to 4 times to obtain the corresponding Solid Support of 3’MVIP.

The general formula of 5'MVIP phosphoramidite monomer is as follows:

When n is 1-4, the linker B in the general formula is branched 1 to 4 times to obtain the corresponding 5"MVIP phosphoramidite monomer.

The following are only exemplary examples of several chemical synthesis processes of 3'MVIP Solid Support and 5'MVIP phosphoramidite monomers. With reference to the methods described in the examples, those skilled in the art can easily synthesize the remaining 3'MVIP Solid Support and 5'MVIP phosphoramidite monomers involved in the present invention. The synthesis process is described as follows:

3.1 Synthesis of Solid Support for 3’MVIP

3.1.1 Synthesis of Solid Support of 3'MVIP09

3'MVIP09's Solid Support

Description of the synthesis process:

3.1.1.1 Synthesis of ERC-01-c1

Weigh 2-amino-1,3-propanediol (5.0 g, 54.9 mmol), add DMSO 50 mL, sodium hydroxide solution (1 g/mL) 5 mL, cool to 0 ° C, add tert-butyl acrylate (20 mL, 137.8 mol) dropwise, add for 2 hours, react at room temperature for 48 hours, add petroleum ether (100 mL), wash twice with saturated brine, and dry the organic layer. Pass through a chromatography column (eluent: ethyl acetate: petroleum ether = 25%-75%), add 0.05% triethylamine to the column, and obtain 6.2 g of colorless oil.

3.1.1.2 Synthesis of ERC-01-c2

Weigh ERC-01-c1 (6.2 g, 17.9 mmol), add 50 mL of dichloromethane and 23 mL of sodium carbonate solution (25%), add benzyl chloroformate (8.2 mL, 57.4 mmol) dropwise at room temperature for 2 hours, react at room temperature overnight, wash 3 times with saturated brine, dry over anhydrous sodium sulfate, evaporate the solvent, and pass through a chromatography column (ethyl acetate: petroleum ether = 5%-30%) to obtain 4.0 g of an oily substance.

3.1.1.3 Synthesis of ERC-01-c3

Take ERC-01-c2 (4.0 g, 8.3 mmol), add 12 mL of formic acid, react at room temperature overnight, evaporate the solvent under reduced pressure to obtain 2.8 g of the product.

3.1.1.4 Synthesis of ERCd-01-c1

Compound ERC-01-c3 (1.11 g, 3.0 mmol) and dlSANC-c4 (3.6 g, 8.04 mmol) were added to DMF (60 mL), followed by HOBt (2.24 g) and HBTU (3.36 g), and then DIEA (4.16 mL) was slowly added. The reaction solution was stirred at room temperature for 3 hours. Water was then added, and the aqueous layer was extracted with dichloromethane (2x10 mL). The organic layers were combined and then washed with saturated sodium bicarbonate (80 mL), water (2x60 mL), and saturated brine (60 mL) in sequence. Dry with anhydrous sodium sulfate, evaporate to dryness under reduced pressure, and purify with silica gel column chromatography (eluent: 3-15% MeOH in DCM). 3.24 g of light yellow solid was obtained.

3.1.1.5 Synthesis of ERCd-01-c2

ERCd-01-c1 (3.24 g, 2.6 mmol) was dissolved in methanol (60 mL), and 10% palladium carbon (0.3 g) and acetic acid (2.0 mL) were added. Then hydrogenation was added under normal pressure and the reaction was allowed to proceed overnight. The reaction solution was filtered through diatomaceous earth, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.9 g of an oily substance ERCd-01-c2, the high-resolution mass spectrum of which is shown in Figure 1.

3.1.1.6 Synthesis of 3'MVIP09-c1

SANCd-01-c0 (0.824 g, 1.5 mmol) and ERCd-01-c2 (1.09 g, 1.0 mmol) were added to the reaction bottle in sequence, and then 10 mL of DCM was added, stirred to dissolve, and TBTU (0.963 g) and DIPEA (0.517 g) were added in sequence. The reaction was allowed to proceed overnight, water was added, and the mixture was extracted with DCM. The organic phase was then washed with saturated brine, dried, filtered, and concentrated, and finally purified by silica gel column to obtain 1.3 g of the product.

3.1.1.7 Synthesis of 3'MVIP09-c2

To the reaction bottle, 3'MVIP09-c1 (1.62 g, 1 μmol) and 10 mL of DCM were added in sequence, and the mixture was dissolved by stirring at room temperature. DMAP (0.366 g) and succinic anhydride (0.2 g, 3 μmol) were added in sequence, and the mixture was reacted by stirring at room temperature. After TLC analysis, if the reaction was qualified, DCM was concentrated, water was added, and the mixture was extracted with DCM. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated, and finally purified by silica gel column to obtain 1.55 g of the product.

3.1.1.8 Solid Support Synthesis of 3'MVIP09

To the reaction bottle, 3’MVIP09-c2 (0.86 g, 0.5 μmol) and 10 mL DMF were added in sequence to dissolve, and then HBTU (0.19 g), DIPEA (0.194 g) and macroporous aminomethyl resin (2.0 g) were added in sequence. The mixture was shaken for 24 h and filtered. The resin was washed with 10% methanol/DCM and capped with 25% acetic acid/pyridine. The degree of substitution was 150 μmol/g.

3.1.2 Synthesis of Solid Support for 3'MVIP17

3'MVIP17 Solid Support

3.1.2.1 Synthesis of SANC-01-c1

The synthesis steps refer to 3.1.1.1. Synthesis of ERC-01-c1.

3.1.2.2 Synthesis of SANC-01-c2

The synthesis steps refer to 3.1.1.2. Synthesis of ERC-01-c2.

3.1.2.3 Synthesis of SANC-01-c3

The synthesis steps refer to 3.1.1.3. Synthesis of ERC-01-c3.

3.1.2.4 Synthesis of SANCd-01-c1

The synthesis steps refer to 3.1.1.4. Synthesis of ERCd-01-c1.

3.1.2.5 Synthesis of SANCd-01-c2

The synthesis steps refer to 3.1.1.5. Synthesis of ERCd-01-c2.

3.1.2.6 Synthesis of 3'MVIP17-c1

The synthesis steps refer to the synthesis of 3’MVIP09-c1 in 3.1.1.6., and the high-resolution mass spectrum of the synthesized 3’MVIP17-c1 is shown in Figure 2.

3.1.2.7 Synthesis of 3'MVIP17-c2

The synthesis steps refer to 3.1.1.7 Synthesis of 3’MVIP09-c2.

3.1.2.8 Solid Support Synthesis of 3'MVIP17

The synthesis steps refer to 3.1.1.8 Solid Support synthesis of 3’MVIP09.

3.1.3 Synthesis of Solid Support of 3'MVIP01:

3'MVIP01 Solid Support

Description of the synthesis process:

3.1.3.1 Synthesis of 3'MVIP01-c1

The synthesis steps refer to the synthesis of 3.1.1.6.3’MVIP09-c1.

3.1.3.2 Synthesis of 3'MVIP01-c2

The synthesis steps refer to the synthesis of 3.1.1.7.3’MVIP09-c2.

3.1.3.3 Solid Support Synthesis of 3'MVIP01

The synthesis steps refer to 3.1.1.8.3’MVIP09’s Solid Support synthesis.

3.2 Synthesis of 5’MVIP phosphoramidite monomer

3.2.1 Synthesis of 5'MVIP09 phosphoramidite monomer:

5'MVIP09 phosphoramidite monomer

3.2.1.1 Synthesis of 5'MVIP09-ERCd-PFP-c1

Weigh ERCd-01-c2 (2.18 g, 2.0 mmol) and dissolve it in DMF (50 mL). Add benzyl glutarate (0.53 g, 2.4 mmol), DIPEA (0.78 g) and TBTU (0.84 g). Stir at room temperature overnight, quench with water (50 mL), extract with DCM (30 mL*3), wash with 10% citric acid (50 mL*3), saturated sodium bicarbonate 50 mL and pyridine 100 mL, dry over anhydrous sodium sulfate, filter, rotary evaporate, and purify by column to obtain the product 5'MVIP09-ERCd-PFP-c1 (2.15 g).

3.2.1.2 Synthesis of 5'MVIP09-ERCd-PFP-c2

Weigh 5'MVIP09-ERCd-PFP-c1 (2.15 g, 1.66 mmol) and 10% palladium carbon (0.21 g), add methanol (50 mL), stir and hydrogenate at room temperature overnight. After the reaction, filter the palladium carbon with diatomaceous earth and rotary evaporate to obtain the crude product of 5'MVIP09-ERCd-PFP-c2 (1.9 g). Its high-resolution mass spectrum is shown in Figure 3.

3.2.1.3 Synthesis of 5'MVIP09-ERCd-PFP

Weigh the crude product of 5'MVIP09-ERCd-PFP-c2 (1.9 g, 1.58 mmol) and dissolve it in DCM (60 mL). Add DIPEA (1.33 g), cool, add pentafluorophenol trifluoroacetate (2.21 g, 7.9 mmol), stir at room temperature for 2 h, then evaporate it by rotary evaporation, dissolve it in DCM (60 mL), wash it with saturated sodium bicarbonate (30 mL*3), 10% citric acid (30 mL*1), and saturated brine (50 mL*1), dry it with anhydrous sodium sulfate, filter it, and evaporate it by rotary evaporation to obtain the crude product of 5'MVIP09-ERCd-PFP (2.35 g). After drying, use it directly in the next reaction without purification.

3.2.1.4 Synthesis of 5'MVIP09 phosphoramidite monomer-c1

The crude product of 5'MVIP09-ERCd-PFP (2.35 g, 1.58 mmol) was dissolved in DCM (60 mL), and DIPEA (0.82 g, 6.32 mmol) and 6-amino-1-hexanol (0.37 g, 3.16 mmol) were added, and the mixture was stirred at room temperature overnight. 10% citric acid (30 mL) was added, and the mixture was extracted with DCM (30 mL*3), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, evaporated, and purified by column to obtain the product 5'MVIP09 monomer-c1 (1.73 g).

3.2.1.5 5'MVIP09 phosphoramidite monomer

Weigh 5'MVIP09 phosphoramidite monomer-c1 (1.3 g, 1.0 mmol) and dissolve it in acetonitrile (30 mL), add diisopropylamine triazole (0.22 g), add bis-(diisopropylamino)(2-cyanoethoxy)phosphine (0.36 g, 1.2 mmol) dropwise under ice bath, react at room temperature for 4 hours, control by HPLC, after the reaction is qualified, concentrate and purify by column to obtain the product 5'MVIP09 monomer (1.2 g).

3.2.2 Synthesis of 5'MVIP01 phosphoramidite monomer:

5'MVIP01 phosphoramidite monomer

Phosphoramidite monomer of 5'MVIP01 Weigh YICd-01-c2 (1.12 g, 2.0 mmol), and refer to 3.2.1.1. to 3.2.1.5 for the remaining operations.

Example 4 Synthesis of RNA Inhibitors Coupled with Carriers

Synthesis description of the antisense strand of the coupling carrier (3’MVIP 09 coupling): Purge the reagent bottle with argon for at least 2 minutes. Add phosphoramidite monomer or and acetonitrile to the reagent bottle in sequence, tighten the bottle cap, and shake until the solid is visually dissolved. Then add 3A molecular sieves and let it stand for more than 8 hours for use. Purge the reagent bottle with argon for at least 2 minutes. Add hydrogenated xanthan and dry pyridine to the reagent bottle in sequence, tighten the bottle cap, and shake until the solid is visually dissolved, and store it for use. Confirm that the following operations are performed at room temperature 20-30℃: Weigh 3’MVIP Solid Support, add it to the reagent bottle, then add acetonitrile, shake and mix evenly, transfer it to the synthesis column, and use acetonitrile to elute the remaining part in the reagent bottle and transfer it to the synthesis column. After the elution is completed, add acetonitrile to fill the synthesis column and record the amount of acetonitrile used. Install and fix the synthesis column according to the instrument operation.

Connect the prepared monomer solution, CAP A, CAP B, oxidant, thiolation agent, activator, decapping agent and acetonitrile to the corresponding pipelines of AKTA PILOT100, ensuring that the pipelines are inserted into the bottom of the reagent bottle.

After the synthesis method is set up, the instrument is ready to work. Click Run to start the synthesis. Observe and record the area of each detritylation peak online. During the synthesis process, add the deprotection reagent according to the actual amount used.

After the synthesis is completed, argon is purged into the synthesis column for ≥2h, and the synthesis column is unloaded according to the operating procedures. The solid phase carrier in the synthesis column is transferred to the reaction bottle, methylamine aqueous solution and ammonia water are added, and the reaction bottle is placed in a shaker at 35°C for 2-3 hours. The solution is filtered into a round-bottom flask, and the residual solid phase is washed with a 50% ethanol aqueous solution, filtered again and combined with the previous filtrate, the round-bottom flask is connected to a rotary evaporator, the water temperature is set to 50°C and evaporated until no distillation, ethanol is added to the round-bottom flask, mixed, and evaporated again until no distillation, and the operation is repeated until white powder appears at the bottom of the bottle. The obtained white powder is prepared into a solution, purified using a reverse chromatography column, and sampled to detect OD260 and purity. The purified antisense chain solution is divided into a syringe bottle and freeze-dried for standby use, and the product is sealed and stored in a -20°C refrigerator.

The synthesis of the sense strand (5’MVIP09 coupling) of the coupled vector is the same as the antisense strand, where the Solid Support loaded on the column is the Universal vector. Add DIPEA to the intermediate to make a solution, add 5’MVIP phosphoramidite monomer, mix well, and place the reaction bottle in a shaker at 35°C for 2-3 hours.

Description of the synthetic annealing process of RNA inhibitors:

Take the obtained positive and antisense strands, mix them in a reaction bottle in an equimolar ratio of 1:1, and after 5 minutes in a water bath at 95°C, turn off the power of the water bath and let it cool naturally to below 40°C. Add 3M sodium acetate aqueous solution to the double-stranded solution, mix evenly, then add an appropriate volume of anhydrous ethanol, mix evenly, and put the reaction solution in a -20°C refrigerator for 45 minutes. Set the refrigerated high-speed centrifuge to 4°C for pre-cooling. After the temperature is reached, put in the double-stranded solution and start the centrifuge. Take out the double-stranded solution after centrifugation, remove the supernatant, add ultrapure water to completely dissolve the solid, take samples to test OD260 and purity, and obtain the RNA inhibitors in Table 14. Dispense the purified finished solution into vials for freeze-drying for later use, and seal the product and store it in a -20°C refrigerator.

The above examples only exemplify the synthesis of RNA inhibitors coupled with 5'MVIP09/3'MVIP09. The RNA inhibitors described in the present invention but not listed here are all applicable to this rule, that is, when the 3' end of the sense chain or antisense chain of the RNA inhibitor is coupled with the carrier structure 3'MVIP, the solid support of 3'MVIP is used as the starting monomer for solid phase synthesis; when the 5' end of the sense chain or antisense chain of the RNA inhibitor is coupled with the carrier structure 5'MVIP, the 5'MVIP phosphoramidite monomer is used as the last monomer for solid phase synthesis. With reference to the method described in this embodiment, those skilled in the art can easily synthesize the remaining RNA inhibitors involved in the present invention.

Example 5 Screening experiment of CFB inhibitor and C5 inhibitor in PHH cells:

The RNA inhibitors of this example are selected from Tables 1-7, and the inhibitory effects of the RNA inhibitors are verified by free uptake by PHHs cells.

Verification method: Day 0, plate PHHs cells and use RNAiMAX transfected the compound into cells. 48 hours after transfection, Total RNA was extracted using the Super Total RNA Extraction Kit (Promega-LS1040), and cDNA was synthesized using One-Step gDNA Removal. Green qPCR SuperMix (+Dye I) was used for qPCR detection.

Data analysis: The target gene mRNA expression level in the sample was calculated based on the Ct value of each sample, and the calculation was performed by the ΔΔCt relative quantitative method. The relative expression of the target gene was expressed by 2-ΔΔCT. The calculation formula is as follows: ΔCT = average Ct value of the target gene - average Ct value of the reference gene; ΔΔCT = ΔCT (drug group) - ΔCT (control group); relative expression of the target gene mRNA = 2-ΔΔCT; target gene inhibition rate = (1-value of sample/mean value of RNAiMAX Control) × 100%, and the results are expressed as mean ± SD of three replicate wells. The inhibitory effect of RNA inhibitors on the complement system mRNA of PHH cells at concentrations of 500nM, 100nM, 10nM, 1nM, 0.02nM, 10nM, 1nM and 0.1nM was investigated. The experimental results of the inhibitory effect of CFB RNA inhibitors on PHH cells are shown in Table 16-1.

Table 16-1 Inhibitory effect of CFB RNA inhibitor on PHH cells

Example 6 In vitro study on inhibition of complement system gene expression by CFB, C5, and C3 modified sequence RNA inhibitors

The RNA inhibitors of this example are selected from Table 7.

Example 6-1 RNA inhibitors were prepared by the method described in Example 4. Referring to the test method of Example 2, the inhibitory effects of RNA inhibitors on CFB mRNA, C5 mRNA, and C3 mRNA in Hep3B cells at concentrations of 1 nM and 0.01 nM were investigated. The test results are shown in Tables 17-1 to 17-5.

Table 17-1 Inhibitory effect of RNA inhibitors on C5 mRNA in Hep3B cells

Table 17-2 Inhibitory effect of RNA inhibitors on C5 mRNA in Hep3B cells

Table 17-3 Inhibitory effect of RNA inhibitors on C5 mRNA in Hep3B cells

Table 17-4 Inhibitory effect of RNA inhibitors on C3 mRNA in Hep3B cells in vitro

Example 6-2 Experimental study on the determination of EC50 of RNA inhibitors for inhibiting C3 gene in vitro

The RNA inhibitors of this embodiment are selected from Table 7. The RNA inhibitors Kylo-27-DS8201, Kylo-27-DS8771 and Kylo-27-DS8141 were investigated for their inhibitory effects on C3 mRNA in Hep3B cells at different concentrations. Three inhibition rate data were tested for each concentration and the average was taken. The inhibition rate-concentration curve was drawn based on the experimental data, and the corresponding EC50, EC75, EC85 and EC95 were calculated. The obtained test results are shown in Tables 18-1 and 18-2.

Table 18-1 Inhibitory effect of RNA inhibitors on C3 mRNA in Hep3B cells

Table 18-2 EC values of RNA inhibitors for C3 mRNA inhibition in Hep3B cells

Example 7 Evaluation of the in vivo activity of RNA inhibitors using mice

The RNA inhibitors of this example are selected from Table 7, and the inhibitory effects of the RNA inhibitors are verified by humanized CFB mice.

Example 7-1: Inhibitory effect of Kylo-17 inhibitor on protein levels in humanized CFB mice

After the experimental animals were adapted to feeding, they were randomly divided into normal saline group, Kylo-17-DS2761, Kylo-17-DS2821, Kylo-17-DS2811, Kylo-17-DS2751, and Kylo-17-DS2861 groups according to the hCFB protein content in plasma on Day-1. There were 5 animals in each group. The drug was administered by a single subcutaneous injection, with a dose of 3 mg/kg, a volume of 5 mL/kg, and a concentration of 0.6 mg/mL. The day of administration was recorded as Day 0. The animals were individually identified by ear tags. The cages were identified by hanging cage cards.

Detection time: About 200 μL of blood was collected from the inner canthus of the eye on Day 1 before administration, Day 7, Day 14, Day 21, Day 28, Day 35 and Day 42 after administration. The whole blood sample was placed in a 37°C water bath for 1 hour before centrifugation, and centrifuged at 3000r for 10 minutes. The supernatant was collected as the fresh serum sample for hCFB protein detection.

Method for detecting hCFB protein level: ELISA kit was used for detection (single well); serum samples were obtained by centrifugation of whole blood samples and tested freshly.

The experimental data were expressed as mean ± standard deviation (Mean ± SD), and converted into the inhibitor effect on the mouse hCFB protein inhibition rate (%, Mean ± SD). T test was used for data statistical analysis, and P < 0.05 indicated statistical significance. The experimental results are shown in Table 19 and Figure 4:

Table 19 Effect of hCFB administration on hCFB protein inhibition rate in mouse serum (%, Mean ± SD) Note: Compared with the saline group, "*" indicates P ≤ 0.05, "**" indicates P ≤ 0.01, and "***" indicates P ≤ 0.001

Example 7-2: Inhibitory effect of Kylo-19 inhibitor on protein levels in humanized C5 mice

After the experimental animals were adapted to feeding, they were randomly divided into saline group, Kylo-19-DS7511, Kylo-19-DS7901, Kylo-19-DS7891, Kylo-19-DS7921, Kylo-19-DS8001 group according to the hC5 protein content in the serum on Day-3, with 5 mice in each group. The drug was administered by a single subcutaneous injection, with a dose of 3 mg/kg, a volume of 5 mL/kg, and a concentration of 0.6 mg/mL. The day of administration was recorded as Day 0. The animals were individually identified by ear tags. The cages were identified by hanging cage cards.

Detection time: About 200 μL of blood was collected from the inner canthus of the eye on Day 3 before administration, Day 7, Day 14, Day 21, Day 28, Day 35, Day 42 and Day 49 after administration. The whole blood sample was placed in a 37°C water bath for 1 hour before centrifugation, and centrifuged at 3000r for 10 minutes. The supernatant was collected as the fresh serum sample for hC5 protein detection.

The experimental data were expressed as mean ± standard deviation (Mean ± SD), and converted into the inhibitor effect on the mouse hC5 protein inhibition rate (%, Mean ± SD). T test was used for data statistical analysis, and P < 0.05 indicated statistical significance. The experimental results are shown in Table 20 and Figure 5:

Table 20 Effect of hC5 administration on the inhibition rate of hC5 protein in mouse serum (%, Mean ± SD) Note: Compared with the normal control group, "*" indicates P≤0.05, "**" indicates P≤0.01, and "***" indicates P≤0.001

Example 7-3: Inhibitory effect of Kylo-19 inhibitor on protein levels in humanized C5 mice

After the experimental animals were adapted to feeding, they were randomly divided into saline group, Kylo-19-DS7871 group, and Kylo-19-DS7881 group according to the hC5 protein content in the serum on Day-3, with 5 mice in each group. The drug was administered by a single subcutaneous injection, with a dose of 3 mg/kg, a volume of 5 mL/kg, and a concentration of 0.6 mg/mL. The day of administration was recorded as Day 0. The animals were individually identified by ear tags. The cages were identified by hanging cage cards.

Detection time: About 200 μL of blood was collected from the inner canthus of the eye on Day 3 before administration, Day 7, Day 14, Day 21, Day 28, Day 35, Day 42 and Day 49 after administration. The whole blood sample was placed in a 37°C water bath for 1 hour before centrifugation, and centrifuged at 3000r for 10 minutes. The supernatant was collected as the fresh serum sample for hC5 protein detection.

The experimental data were expressed as mean ± standard deviation (Mean ± SD), and converted into the inhibitor effect on the mouse hC5 protein inhibition rate (%, Mean ± SD). T test was used for data statistical analysis, and P < 0.05 indicated statistical significance. The experimental results are shown in Table 21 and Figure 6:

Table 21 Effect of hC5 administration on the hC5 protein inhibition rate in mouse serum (%, Mean ± SD) Note: Compared with the normal control group, "*" indicates P≤0.05, "**" indicates P≤0.01, and "***" indicates P≤0.001

Inhibitors with significant inhibitory effects were selected and optimized for cynomolgus monkey experiments.

Example 8 Evaluation of the in vivo activity of RNA inhibitors using cynomolgus monkeys

Example 8-1 Validation experiment of the pharmacodynamic activity of CFB inhibitors in NHP cynomolgus monkeys

The RNA inhibitors of this embodiment are selected from Table 7, and the inhibitory effects of the positive reference group and Kylo-17-DS2911 inhibitor on the expression of serum CFB protein level and CFB mRNA level in NHP cynomolgus monkeys are verified by cynomolgus monkeys. The inhibitors selected for the positive reference group are: positive chain: AsAsGAGAfAGfUfCfGUUUCAUUCAU-L96, antisense chain: AsfUsGAAfUGfAfAACGAfCUfUCUCUUsGsU.

After the healthy cynomolgus monkeys were adapted to feeding, blood was collected on Day-1 to detect the CFB protein content in the serum. The animals were grouped according to the CFB protein content, and divided into normal control group, positive reference group, and Kylo-17-DS2911. The dosage was 6 mg/kg, with 2 animals in the normal control group, 1 male and 1 female; 3 animals in each of the positive reference group and G3 group, 2 males and 1 female. A single subcutaneous injection (neck back injection) was administered, with a dosing volume of 1 mL/kg, and the day of administration was recorded as Day 0. On Day-2 before administration, Day 7, Day 14, Day 21, and Day 28 after administration, venous blood was collected once a week until the end of the experiment D91 to detect the CFB protein level in the serum of NHP cynomolgus monkeys.

CFB protein level detection method: ELISA kit detection (single well); serum samples are freshly tested after centrifugation of whole blood samples. Detection time: Day-1 before administration, Day 7, Day 14, Day 21, Day 28 after administration, and venous blood collection once a week until the end of the test. About 2mL of blood is collected each time. The whole blood sample is left at room temperature for 1 hour before centrifugation, centrifuged at 3000r for 10 minutes, and the supernatant is collected, which is the freshly obtained serum sample for CFB protein detection.

Method for detecting CFB mRNA levels in liver tissue: Liver biopsy was performed on crab-eating macaques, and the mRNA level of the target in the liver was determined using the probe-based qPCR method.

The experimental data were expressed as mean ± standard deviation (Mean ± SD), and GraphPad Prism 8.3 analysis software was used for data plotting and analysis. T test was used for data statistical analysis, and P < 0.05 indicated statistical significance.

The results of the CFB protein level test are shown in Table 23 and Figure 8; the results of the CFB mRNA level test are shown in Table 24 and Figure 9:

Table 23 Effect of CFB RNA inhibitor on the inhibition rate of CFB protein in NHP cynomolgus monkeys (%, Mean ± SD)

Table 23

Table 24 Effects of CFB RNA inhibitor on CFB mRNA levels in liver tissue of cynomolgus monkeys

From the test results in Table 23, it can be seen that in terms of CFB protein level, the inhibition rate rose rapidly on D28 after administration, and the CFB protein level could be continuously inhibited from D28 to D63 (the inhibition rate ranged from 78.8% to 89.52%, P<0.05 or P<0.01 or P<0.001), and the inhibition rate ranged from D70 to D91 from 64.23% to 72.18%. From D21, the inhibition rate of Kylo-17-DS2911 surpassed that of Yangshen, showing a significant inhibitory effect, and from the inhibition rate of D21 to D91, it can be seen that the inhibitory effect is stable. From the inhibition rate of Kylo-17-DS2911 compared with Yangshen on D70-D91, it can be seen that the inhibitor of the present invention has a more stable inhibitory effect.

From the test results in Table 24, we can see that: in terms of CFB mRNA levels in liver tissue: Kylo-17-DS2911 significantly decreased CFB mRNA levels in liver tissue at D35 after administration, with an inhibition rate of 96.5%, and it can still stably and efficiently inhibit CFB mRNA expression at D67 and D84, with inhibition rates of 89.66% and 89.17%, respectively. Compared with Yangshen, it has a more significant CFB mRNA inhibition level, and the inhibition is more stable and lasting.

Example 8-2 Effect of C5 inhibitor on C5 protein content and CH50 activity in serum of NHP cynomolgus monkeys

The RNA inhibitors of this embodiment are selected from Table 7, and the positive reference group (sense chain: AsAsfGCfAAfGAfUfAfUUfUUUfAUfAAUA-L96, antisense chain: UsfAsfUUfAUAfAAfAAUAfUCfUUfGCUUsUsUdTdT) and the effects of the Kylo-19-DS7881 inhibitor on serum C5 protein content and CH50 activity in NHP crab-eating monkeys are verified by crab-eating monkeys.

After the healthy cynomolgus monkeys were adapted to feeding, blood was collected on Day-2 to detect the C5 protein content in the serum. The animals were grouped according to the C5 protein content, and divided into normal control group G1, positive reference group G2, and Kylo-19-DS7881G3. The dosage was 6 mg/kg, with 2 animals in the normal control group, 1 male and 1 female; 3 animals in each group of G2 and G3, 2 females and 1 male. A single subcutaneous injection (neck back injection) was administered, the administration volume was 1 mL/kg, and the day of administration was recorded as Day 0. On Day-2 before administration, Day 7, Day 14, Day 21, and Day 28 after administration, venous blood was collected once a week until the end of the experiment D91 to detect the C5 protein level in the serum of NHP cynomolgus monkeys.

On Day 1 before administration, Day 7, Day 14, Day 21, and Day 28 after administration, venous blood was collected once a week until the end of the experiment. About 2 mL of blood was collected each time. The whole blood sample was allowed to stand at room temperature for 1 hour before centrifugation, and centrifuged at 3000r for 10 minutes. The supernatant was collected, which was the fresh serum sample for C5 protein detection. C5 protein level detection method: ELISA kit detection (single well); serum samples were freshly tested after whole blood samples were centrifuged.

Detect CH50 activity in serum samples on Day 2 before administration, Day 14, Day 28, Day 42, Day 49, Day 56, Day 63, Day 70, and Day 77 after administration. CH50 activity detection method: ELISA kit detection (single well); serum samples are obtained after centrifugation of whole blood samples.

The experimental data were expressed as mean ± standard deviation (Mean ± SD), and GraphPad Prism 8.3 analysis software was used for data plotting and analysis. T test was used for data statistical analysis, and P < 0.05 indicated statistical significance.

The results of C5 protein inhibition rate are shown in Table 24 and Figure 10; the results of CH50 activity level test are shown in Table 25 and Figure 11:

Table 24 Effect of C5 RNA inhibitor on the inhibition rate of C5 protein in NHP cynomolgus monkeys (%, Mean ± SD) Note: Compared with the normal control group, "*" indicates P≤0.05, "**" indicates P≤0.01, and "***" indicates P≤0.001

Table 24 Note: Compared with the normal control group, "*" indicates P≤0.05, "**" indicates P≤0.01, and "***" indicates P≤0.001

Table 25 Effect of C5 RNA inhibitor on the retention level of CH50 activity in NHP cynomolgus monkeys (Mean±SD)

Table 25

From Table 24 and Figure 10, it can be seen that the serum C5 protein level of Kylo-19-DS7881 of the present invention decreased significantly on D7 after administration, with an inhibition rate of 71.4%, and further decreased on D14. The expression of serum C5 protein level was continuously and stably inhibited during D14-D77 (the inhibition rate range was 89.2%-100%, P<0.05or P<0.01or P<0.001), and the protein level on D84 rebounded after administration, with inhibition rates of 83.4% and 79.4% on D84 and D91, respectively. Compared with the positive reference, the inhibition ability was significantly better after D56 days, and it was more stable and the inhibition effect lasted longer than the positive reference.

It can be seen from Table 25 and Figure 11 that the serum CH50 activity of Kylo-19-DS7881 of the present invention decreased significantly on D14 after administration, and the CH50 activity retention level was 35%. The serum CH50 activity continued to decrease steadily during D28-D63 (the retention levels ranged from 38.8% to 40.3%, respectively, P<0.05). The CH50 activity rebounded on D70 after administration, and the CH50 activity retention levels at D70 and D77 were 48.7% and 58.4%, respectively. Compared with the positive reference, the effect of reducing CH50 activity is better, and the reducing effect is more persistent.

Example 8-3 Effect of C3 inhibitor on C3 protein content in serum of NHP cynomolgus monkeys

The RNA inhibitors of this example are selected from Table 7, and the inhibitory effects of Kylo-27-DS8141 and Kylo-27-DS8201 inhibitors on the expression of serum C3 protein levels in NHP cynomolgus monkeys were evaluated.

After healthy cynomolgus monkeys were adapted to feeding, blood was collected on Day-1 to detect the C3 protein content in serum. The animals were grouped according to the C3 protein content, and divided into normal control group, Kylo-27-DS8141, Kylo-27-DS8131, Kylo-27-DS8201, and Kylo-27-DS8211. The dosage was 6 mg/kg, with 3 animals in each group, 2 males and 1 female. A single subcutaneous injection (neck back injection) was administered, with a volume of 1 mL/kg, and the day of administration was recorded as Day 0.

On Day 1 before administration, Day 7, Day 14, Day 21, Day 28, Day 35, and Day 42 after administration, venous blood was collected once a week until the end of the experiment. About 2 mL of blood was collected each time. The whole blood sample was left at room temperature for 1 hour before centrifugation, and centrifuged at 3000r for 10 minutes. The supernatant was collected as the fresh serum sample for C3 protein detection.

C3 protein level detection method: ELISA kit detection (single well); serum samples are freshly tested after centrifugation of whole blood samples.

The experimental data were expressed as mean ± standard deviation (Mean ± SD), and GraphPad Prism 8.3 analysis software was used for data plotting and analysis. T test was used for data statistical analysis, and P < 0.05 indicated statistical significance. The experimental results are shown in Table 26 and Figure 12.

Table 26 Effect of C3 RNA inhibitor on the inhibition rate of C3 protein in NHP cynomolgus monkeys (%, Mean ± SD)

Table 26 Note: Compared with the normal control group, "*" indicates P≤0.05, "**" indicates P≤0.01, and "***" indicates P≤0.001

[Corrected 20.01.2025 in accordance with Article 91]
As shown in Table 26 and Figure 12, the C3 protein inhibition rates of Kylo-27-DS8141 and Kylo-27-DS8201 are more significant and persistent. Kylo-27-DS8141 continuously and stably inhibits the expression of serum C3 protein levels during D14-D70 (the inhibition rate range is 79.6%-91.6%, P<0.05or P<0.01), and the inhibition rates can be maintained at 74.3% and 67.9% at D77 and D84, respectively, with good persistence. Kylo-27-DS8201 continuously and stably inhibits the expression of serum C3 protein levels during D14-D98, with the inhibition rate reaching 96.8% at D42 and still maintaining 85.1% at D98, with good persistence.

Claims (34)

An RNA inhibitor for inhibiting complement system gene expression or a pharmaceutically acceptable salt thereof, characterized in that the RNA inhibitor is formed by base pairing of a sense strand and an antisense strand with a chain length of 15-30, preferably 19-23, wherein the antisense strand includes a region complementary to an mRNA encoding a complement system, and wherein the complementary region comprises at least 15 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides from any antisense strand in any of Tables 1-1, 1-2, and 1-3. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1 is characterized in that the RNA inhibitor is an RNA inhibitor that inhibits CFB gene expression, wherein the antisense strand includes a region complementary to a target sequence, and the target sequence is: 5'gucuagucaacuuaauugaga 3' SEQ ID NO: 25, the starting position in NM_001710.5 is at 1157, and there is at least 85% base complementarity between the sense strand and the antisense strand. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1 is characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C5 gene expression, wherein the antisense strand includes a region complementary to a target sequence, and the target sequence is: 5'uugucccaguauucuauguuu 3' SEQ ID NO: 826, and the starting position in NM_001735.3 is at position 3073, and there is at least 85% base complementarity between the sense strand and the antisense strand. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C3 gene expression, wherein the antisense strand includes a region complementary to a target sequence, and the target sequence is: 5'gguguugacagauacaucu 3' SEQ ID NO: 1952, the starting position in NM_000064.4 is at position 4329; 5'ggagccuacagagaaauucua 3' SEQ ID NO: 2039, the starting position in NM_000064.4 is at position 771; the target sequence is: 5'agaaau ucuacuacaucuaua 3' SEQ ID NO: 2048, the starting position in NM_000064.4 is 782; the target sequence is: 5'gcugaggagaauugcuucaua 3' SEQ ID NO: 2240, the starting position in NM_000064.4 is 4603; the target sequence is: 5'ggagaauugcuucauacaaaa 3' SEQ ID NO: 2245, the starting position in NM_000064.4 is 4608; there is at least 85% base complementarity between the sense chain and the antisense chain. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that the antisense strand is selected from the following sequences: 5'ucucaauuaaguugacuagacac 3' SEQ ID NO: 293; or a sequence having at least 15 consecutive nucleotides identical to SEQ ID NO: 293, or a sequence differing from SEQ ID NO: 293 by one, two or three nucleotides, Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that the sense strand is selected from the following sequences: 5'gucuagucaacuuaauugaga 3' SEQ ID NO: 25; or a sequence having at least 15 consecutive nucleotides identical to the sequence of SEQ ID NO: 25, or a sequence differing from the sequence of SEQ ID NO: 25 by one, two or three nucleotides, Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that the antisense strand is selected from the following sequences: 5'aaacauagaauacugggacaacg 3' SEQ ID NO: 1278; or a sequence having at least 15 consecutive nucleotides identical to the sequence of SEQ ID NO: 1278, or a sequence differing from the sequence of SEQ ID NO: 1278 by one, two or three nucleotides, Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that the sense strand is selected from the following sequences: 5'uugucccaguauucuauguuu 3' SEQ ID NO: 826; or a sequence having at least 15 consecutive nucleotides identical to the sequence of SEQ ID NO: 826, or a sequence differing from the sequence of SEQ ID NO: 826 by one, two or three nucleotides, Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that the antisense strand is selected from the following sequences: 5'agauguaucugucaacaccau 3' SEQ ID NO.2543; 5'uagaauuucucuguaggcuccac 3' SEQ ID NO.2630; 5'uauagauguaguagaauuucucu 3' SEQ ID NO.2639; 5'uaugaagcaauucuccagcac 3' SEQ ID NO.2831; 5'uuuuguaugaagcaauucuccuc 3' SEQ ID NO.2590; or a sequence having at least 15 consecutive nucleotides identical to the above sequence, or a sequence differing from the above sequence by one, two or three nucleotides, Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that the sense strand is selected from the following sequences: 5'gguguugacagauacaucu 3' SEQ ID NO.1952; 5'ggagccuacagagaaauucua 3' SEQ ID NO.2039; 5'agaaauucuacuacaucuaua 3' SEQ ID NO.2048; 5'gcugaggagaauugcuucaua 3' SEQ ID NO.2240; 5'ggagaauugcuucauacaaaa 3' SEQ ID NO.2245; or a sequence having at least 15 consecutive nucleotides identical to the above sequence, or a sequence differing from the above sequence by one, two or three nucleotides, Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that the sense strand is SEQ ID NO: 25 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 293 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides: Sense strand: 5'gucuagucaacuuaauugaga 3' SEQ ID NO: 25; Antisense strand: 5'ucucaauuaaguugacuagacac 3' SEQ ID NO: 293; Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that the sense strand is SEQ ID NO: 826 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 1278 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides: Sense strand: 5'uugucccaguauucuauguuu 3' SEQ ID NO: 826; Antisense strand: 5'aaacauagaauacugggacaacg 3' SEQ ID NO: 1278; Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that the sense strand is SEQ ID NO: 2048 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 2639 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides: Sense strand: 5'agaaauucuacuacaucuaua 3' SEQ ID NO: 2048; Antisense strand: 5'uauagauguaguagaauuucucu 3' SEQ ID NO: 2639; Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that the sense strand is SEQ ID NO: 2639 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides; and the antisense strand is SEQ ID NO: 2831 or a sequence having at least 15 consecutive nucleotides identical thereto, or a sequence differing therefrom by one, two or three nucleotides: Sense strand: 5'gcugaggagaauugcuucaua 3' SEQ ID NO: 2240; Antisense strand: 5'uaugaagcaauucuccucagcac 3' SEQ ID NO: 2831; Among them, g = guanylate, a = adenylate, u = uridylate, c = cytidine, t = thymidine deoxyribonucleotide. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to any one of claim 1, characterized in that at least one nucleotide of the RNA inhibitor is modified. The RNA inhibitor or pharmaceutically acceptable salt thereof according to claim 15, characterized in that the modification comprises: 2'-fluoro modification, 2'-methoxy modification, phosphorothioate modification, invAb modification, glycerol nucleotide, 3' terminal deoxythymine (dT) nucleotide, locked nucleotide, unlocked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxy modified nucleotide, 2'- The invention can include one or more combinations of methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-phosphate-modified or 2-O-(N-methylacetamide)-modified nucleotides, morpholino nucleotides, aminophosphorylates, abasic nucleotides, abasic deoxynucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, methylphosphonate-modified nucleotides, 5'-phosphate-modified, 5'-phosphate-analog-modified, thermally unstable nucleotides, and nucleotide analogs. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits CFB gene expression, and the antisense strand is selected from the following sequences: 5'UsdCsUCdAATUAAGTUfGACUAGACsAsC 3' SEQ ID NO: 588; Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-fluorouridylate, fC = 2'-fluorocytidine; fGs = 2'-fluoro-3'-thioguanylate, T = 2'-deoxy-thymidylate, dA = 2'-deoxy-adenylate, dG = 2'-deoxy-guanylate. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C5 gene expression, and the antisense strand is selected from the following sequences: 5'AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3' SEQ ID NO: 1598; Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-fluorouridylate, fC = 2'-fluorocytidine; fGs = 2'-fluoro-3'-thioguanylate, T = 2'-deoxy-thymidylate, dA = 2'-deoxy-adenylate, dG = 2'-deoxy-guanylate. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C3 gene expression, and the antisense strand is selected from the following sequences: 5'UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3' SEQ ID NO: 1665; 5'UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3' SEQ ID NO: 1671; Wherein, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, f A = 2'-fluoroadenylic acid, fU = 2'-fluorouridylic acid, fC = 2'-fluorocytidylic acid; fGs = 2'-fluoro-3'-thioguanylic acid, fAs = 2'-fluoro-3'-thioadenylic acid, fUs = 2'-fluoro-3'-thiouridylic acid, fCs = 2'-fluoro-3'-thiocytidylic acid, T = 2'-deoxy-thymidylic acid, dG = 2'-deoxy-guanylic acid, dAs = 2'-deoxy-3'-thioadenylic acid. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits CFB gene expression, and the sense strand is selected from the following sequences: 5’GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3’ SEQ ID NO: 559; Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-fluorouridylate, fC = 2'-fluorocytidine. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C5 gene expression, and the sense strand is selected from the following sequences: 5’UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3’ SEQ ID NO: 1537; Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-fluorouridylate, fC = 2'-fluorocytidine. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C3 gene expression, and the sense strand is selected from the following sequences: 5'AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3' SEQ ID NO: 1645; 5'GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' SEQ ID NO: 1651; Among them, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, fA = 2'-fluoroadenylate, fU = 2'-fluorouridylate, fC = 2'-fluorocytidine. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits CFB gene expression, the sense strand is SEQ ID NO: 559 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 588 or a sequence that differs therefrom by one, two or three nucleotides: Sense strand: 5’GsUsCUAGfUCfAfAfCUUAAUUGAsGsA 3’ SEQ ID NO: 559; Antisense strand: 5'UsdCsUCdAATUAAGTUfGACUAGACsAsC 3' SEQ ID NO: 588; Wherein, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, f A = 2'-fluoroadenylic acid, fU = 2'-fluorouridylic acid, fC = 2'-fluorocytidylic acid; fGs = 2'-fluoro-3'-thioguanylic acid, fAs = 2'-fluoro-3'-thioadenylic acid, fUs = 2'-fluoro-3'-thiouridylic acid, fCs = 2'-fluoro-3'-thiocytidylic acid, T = 2'-deoxy-thymidylic acid, Ts = 2'-deoxy-3'-thiothymidylic acid, dA = 2'-deoxy-adenylic acid. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C5 gene expression, the sense strand is SEQ ID NO: 1537 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 1598 or a sequence that differs therefrom by one, two or three nucleotides: Sense strand: 5’UsUsGUCCfCAfGfUfAUUCUAUGUsUsU 3’ SEQ ID NO: 1537; Antisense strand: 5'AsdAsACdAUdAGAAUdACfUGGGACAAsCsG 3' SEQ ID NO: 1598; Wherein, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, f A = 2'-fluoroadenylic acid, fU = 2'-fluorouridylic acid, fC = 2'-fluorocytidylic acid; fGs = 2'-fluoro-3'-thioguanylic acid, fAs = 2'-fluoro-3'-thioadenylic acid, fUs = 2'-fluoro-3'-thiouridylic acid, fCs = 2'-fluoro-3'-thiocytidylic acid, T = 2'-deoxy-thymidylic acid, Ts = 2'-deoxy-3'-thiothymidylic acid, dA = 2'-deoxy-adenylic acid. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 16, characterized in that the RNA inhibitor is an RNA inhibitor that inhibits C3 gene expression, the sense strand is SEQ ID NO: 1651 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 1671 or a sequence that differs therefrom by one, two or three nucleotides: Sense strand: 5’AsGsAAAUfUCfUfAfCUACAUCUAsUsA 3’ SEQ ID NO: 1645; Antisense strand: 5'UsdAsUAdGATGUAGTAfGAAUUUCUsCsU 3' SEQ ID NO: 1665; Alternatively, the sense strand is SEQ ID NO: 1645 or a sequence that differs therefrom by one, two or three nucleotides, and the antisense strand is SEQ ID NO: 1665 or a sequence that differs therefrom by one, two or three nucleotides: Sense strand: 5'GsCsUGAGfGAfGfAfAUUGCUUCAsUsA 3' SEQ ID NO: 1651; Antisense strand: 5'UsfAsUGAfAGfCAAUUCfUCfCUCAGCsAsC 3' SEQ ID NO：1671; Wherein, G = 2'-O-methylguanylate, A = 2'-O-methyladenylate, U = 2'-O-methyluridylate, C = 2'-O-methylcytidine; Gs = 2'-O-methyl-3'-thioguanylate, As = 2'-O-methyl-3'-thioadenylate, Us = 2'-O-methyl-3'-thiouridylate, Cs = 2'-O-methyl-3'-thiocytidine; fG = 2'-fluoroguanylate, f A = 2'-fluoroadenylic acid, fU = 2'-fluorouridylic acid, fC = 2'-fluorocytidylic acid; fGs = 2'-fluoro-3'-thioguanylic acid, fAs = 2'-fluoro-3'-thioadenylic acid, fUs = 2'-fluoro-3'-thiouridylic acid, fCs = 2'-fluoro-3'-thiocytidylic acid, T = 2'-deoxy-thymidylic acid, dG = 2'-deoxy-guanylic acid, dAs = 2'-deoxy-3'-thioadenylic acid. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, characterized in that the RNA inhibitor further contains carrier structures 5'MVIP and 3'MVIP, and the structure of the RNA inhibitor is as shown in Formula Ia, Ib or Ic:
in, The 5'MVIP is composed of a transfer point R ₁ , a connecting chain D, a linker B, a branch chain L and a liver-targeting specific ligand X, which is connected to the 5' end of the sense chain or the 5' end of the antisense chain through the transfer point R _1. Its structure is shown in general formula I: (XL) _n -BDR ₁ - I The 3'MVIP is composed of a transfer point R ₂ , a connecting chain D, a linker B, a branch chain L and a liver-targeting specific ligand X, which is connected to the 3' end of the sense chain or the 3' end of the antisense chain through the transfer point R _2. Its structure is shown in general formula II: (XL) _m -BDR ₂ - II in, n and m are each independently any integer of 0-4, each independently preferably an integer of 1-3, and n+m=an integer of 2-6, preferably n+m=2, 3 or 4, more preferably 4; The transition point _R1 is a heterocyclic or carbocyclic structure containing N, S or O as shown below:
Alternatively, R ₁ is -NH(CH ₂ ) _x CH ₂ O-, wherein x is any integer from 3 to 12, preferably any integer from 4 to 6; The transition point _R2 is a heterocyclic or carbocyclic structure containing N, S or O as shown below:
Alternatively, the transition point R ₂ is -NH(CH ₂ ) _x1 CH(OH)(CH ₂ ) _x2 CH ₂ O-, wherein x1 is any integer from 1 to 4, and x2 is any integer from 0 to 4; The liver-targeting specific ligand X is the same or different within each of 5'MVIP and 3'MVIP or between 5'MVIP and 3'MVIP, and is selected from monosaccharides and their derivatives, preferably N-acetylgalactosamine and its derivatives, and more preferably selected from the following structures:
Wherein, W is selected from one or two of -OH, -NHCOOH and -NHCO(CH ₂ ) _q CH ₃ , wherein q is an integer of 0-4; The branched chain L is the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP, and is selected from one or more of the following structures:
Wherein, r1 is any integer from 1 to 12, r2 is any integer from 0 to 20, and Z is H, an alkyl group or an amide group, wherein the alkyl group is, for example, a C ₁ -C ₅ alkyl group; The linker B is the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP, and is selected from the following structures:
wherein _A1 and _A2 are each independently C, O, S, -NH-, carbonyl, amide, phosphoryl or thiophosphoryl, and r is any integer from 0 to 4; The connecting chain D is the same or different within each of the 5'MVIP and the 3'MVIP or between the 5'MVIP and the 3'MVIP, and is selected from the following structures:


Wherein, each p is independently any integer from 1 to 20; s is any integer from 2 to 13; _Z1 and _Z2 are the same or different substituent groups. The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 26, characterized in that the 5'MVIP is 5'MVIP01 or 5'MVIP09 as shown below, and the 3'MVIP is 3'MVIP01, 3'MVIP09 or 3'MVIP17 as shown below:
The RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 27, characterized in that the combination of the sense strand 5'MVIP and the antisense strand 3'MVIP is 5'MVIP01/3'MVIP01, 5'MVIP01/3'MVIP17 or 5'MVIP09/3'MVIP09, or the combination of the sense strand 5'MVIP and the antisense strand 3'MVIP is 5'MVIP01/3'MVIP09 or 5'MVIP09/3'MVIP01. The CFB RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the RNA inhibitor is selected from Kylo-17-DS2911. The C5 RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the RNA inhibitor is selected from Kylo-19-DS7881. The C3 RNA inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that the RNA inhibitor is selected from Kylo-27-DS8201 and Kylo-27-DS8141. Use of the RNA inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 31 in the preparation of a medicament for treating and/or preventing a disease associated with an elevated level of the complement system, wherein the disease includes but is not limited to lipid metabolism disorders. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the RNA inhibitor according to any one of claims 1 to 31 or other therapeutic agents for treating or preventing complement system-related diseases. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the RNA inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 31 and a pharmaceutically acceptable excipient, and the dosage form is an oral agent, an intravenous injection, or a subcutaneous or intramuscular injection, preferably a subcutaneous injection.