Oligonucleotide targeting complement component 3 (C3) gene and use thereof

Abstract

Provided are an oligonucleotide targeting a complement component 3 (C3) gene and a use thereof. The oligonucleotide can effectively reduce the content of complement C3 in a body, and is an effective inhibitor of the complement C3.

Description

Oligonucleotides targeting complement component C3 gene and uses thereof Technical Field

[0001] The present disclosure relates to an oligonucleotide, in particular to an oligonucleotide for inhibiting the expression of complement C3 gene and treating diseases associated with abnormal complement expression. Background Art

[0002] The complement system is one of the oldest branches of the immune system, discovered over a century ago. It is called complement because people discovered that in addition to antibodies, there are other proteins in the body that have a supplementary effect on the immune system.

[0003] Complement is a serum protein that primarily mediates immune and inflammatory responses. It can be activated by antigen-antibody complexes or microorganisms, leading to the lysis or phagocytosis of pathogenic microorganisms. The complement system is not only an important effector mechanism for the body's natural immune defense, but also one of the main humoral immune effector mechanisms. When involved in defense, a large amount of complement precursor proteins are rapidly produced to respond to and detect threats, playing an important role in eliminating the invasion of foreign antigens and maintaining the balance of the body's internal environment. Although complement activation is an immune defense response of the host, abnormal overactivation can cause tissue and organ damage, leading to a variety of diseases such as PNH (paroxysmal nocturnal hemoglobinuria), atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, and age-related macular degeneration (AMD). Therefore, targeted complement drugs have broad market prospects.

[0004] The entire activation process of the complement system is manifested as a cascade of enzymatic reactions involving a series of serine proteases, ultimately forming a membrane attack complex that causes target cell damage. Activation of the complement system occurs primarily through three relatively independent yet interconnected pathways: the classical pathway, the alternative pathway, and the lectin pathway (Figure 1). The C5 convertases produced by these three pathways can all cleave C5, triggering a common terminal effect, thereby exerting a variety of biological effects, including regulating phagocytosis, lysing cells, mediating inflammation, immunomodulation, and clearing immune complexes. Currently, drug development targeting targets related to the three pathways has been successfully transformed. Given the clear and specific complement-related pathogenesis of some rare diseases, patients with rare diseases currently benefit the most from them.

[0005] Complement C3 is the most abundant complement component in serum, located upstream of C5. It is the terminal effector molecule in multiple complement activation pathways. Complement C3 is cleaved into C3a and C3b by C3 convertase, playing a crucial role in both the classical and alternative complement activation pathways.

[0006] Complement C5, a representative member of the membrane attack complex (MAC) in the complement system, is a popular target in complement-targeted drug development. C5 inhibitors are among the earliest approved complement drugs. In March 2007, the FDA approved Alexion's C5-targeting monoclonal antibody, eculizumab (Soliris), for the treatment of paroxysmal nocturnal hemoglobinuria (PNH).

[0007] Soliris is the world's first C5 complement inhibitor, administered via biweekly injection of eculizumab. It is also the first specific treatment for patients with paroxysmal nocturnal hemorrhage (PNH). Since then, Soliris has also been approved for atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), and neuromyelitis optica in adults.

[0008] Apellis Pharmaceuticals has developed a complement C3 inhibitor, pegcetacoplan (APL-2). Pegcetacoplan is a synthetic cyclic peptide conjugated to a polyethylene glycol polymer that specifically binds to C3 and C3b. It is currently being developed for the treatment of various diseases, including paroxysmal nocturnal hemoglobinuria (PNH), geographic atrophy (GA), and C3 glomerulopathy.

[0009] Inappropriate activation of the complement system is responsible for the propagation and / or initiation of pathological processes in many different diseases, including, for example, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), C3 glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, ischemia-reperfusion injury, and neurodegenerative diseases. There are limited therapies available for treating diseases associated with complement component C3, which require time-consuming and invasive administration and are costly. Therefore, there is a need in the art for alternative therapies and combination therapies for subjects with diseases associated with complement component C3. Summary of the Invention

[0010] The purpose of the present disclosure is to provide an inhibitor for inhibiting the expression of complement component C3 with good efficacy, high safety and long-lasting efficacy.

[0011] The present disclosure provides oligonucleotides or pharmaceutically acceptable salts thereof and methods of using the oligonucleotides or pharmaceutically acceptable salts thereof to inhibit the expression of complement component C3 gene in cells or mammals, wherein the oligonucleotide targets the complement component C3 gene. Also provided herein are compositions and methods for treating pathological conditions and diseases in mammals caused by the expression of complement component C3 gene. The oligonucleotide is a double-stranded RNA (dsRNA) that directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

[0012] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting complement component C3 gene expression, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having a sequence having at least 80% sequence identity to a sequence shown in any one of SEQ ID NOs. 1-49 and 51-305, or a fragment thereof, or a modified sequence thereof, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; the antisense strand having a sequence having at least 80% sequence identity to a sequence shown in any one of SEQ ID NOs. 306-586, 588-586, and 1002-1009, or a fragment thereof, or a modified sequence thereof, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0013] In another aspect, the present disclosure provides a conjugate for inhibiting the expression of complement component C3 or a pharmaceutically acceptable salt thereof, comprising: (i) an oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to a targeting ligand.

[0014] In another aspect, the present disclosure provides a composition comprising the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, or the aforementioned conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0015] In another aspect, the present disclosure provides use of the aforementioned oligonucleotide or a pharmaceutically acceptable salt, conjugate or a pharmaceutically acceptable salt or composition thereof in the preparation of a medicament for treating and / or preventing a complement component C3-related disorder.

[0016] In another aspect, the present disclosure provides a method for treating and / or preventing complement component C3-associated disorders, disorders and / or conditions in a subject by administering a therapeutic agent (e.g., the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, or the aforementioned conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned composition, or a vector or transgene encoding the oligonucleotide) to the subject.

[0017] In another aspect, the present disclosure provides methods for treating and / or preventing complement C3-associated disorders, disorders and / or conditions in a subject using the aforementioned oligonucleotides or pharmaceutically acceptable salts thereof, conjugates or pharmaceutically acceptable salts thereof, or compositions in combination with other drugs and / or other therapeutic methods.

[0018] Experiments have shown that the oligonucleotide disclosed herein can effectively reduce the level of complement component C3 in the body and is an effective inhibitor of complement component C3. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the complement activation pathway.

[0020] FIG2 shows a flow chart of siRNA solid phase synthesis.

[0021] FIG3 shows the restriction enzyme cleavage map of RA177 pFB-AAV-CAG-Gluc-2A-HsC3_P1.

[0022] FIG4 shows the enzyme cleavage map of RA178 pFB-AAV-CAG-Gluc-2A-HsC3_P2.

[0023] FIG5 shows the in vivo efficacy of hC3 siRNA (AAV hC3 transgenic mice).

[0024] FIG6 shows the in vivo efficacy of hC3 siRNA (hC3 transgenic mice). DETAILED DESCRIPTION

[0025] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0026] As used herein, the term "approximately" or "approximately" as applied to one or more target values ​​refers to a value similar to a reference value. In certain embodiments, unless otherwise indicated or in addition apparent from context, the term "approximately" or "approximately" refers to a value falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) or less (unless such numerals will exceed 100% of possible values).

[0027] The RNAi agents of complement component C3 are described herein for selectively and effectively inhibiting the expression of complement component C3 gene. The RNAi agents of complement component C3 described herein can be used to prevent or treat the following diseases or to prepare medicaments for preventing or treating the following diseases, including but not limited to: cold agglutinin disease (CAD), warm autoimmune hemolytic anemia, and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), bullous pemphigoid, pemphigus, such as pemphigus vulgaris (PV) and pemphigus foliaceus (PF), or C3 glomerulopathy.

[0028] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a complement component C3 gene, including mRNA that is a product of RNA processing of the primary transcript.

[0029] As used herein, term " complementary " refers to the structural relationship that allows nucleotide to form base pairs with each other between nucleotide (for example, on relative nucleic acid or on two nucleotides on the relative region of single nucleic acid chain).For example, the purine nucleotides complementary to the pyrimidine nucleotides of a nucleic acid can be base paired together by forming hydrogen bonds with each other.In some embodiments, complementary nucleotides can be base paired in Watson-Crick (Watson-Crick) mode or in any other manner that allows to form a stable duplex.In some embodiments, two nucleic acids can have and be complementary to each other to form the nucleotide sequence of complementary region, as described herein.

[0030] As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, e.g., a 5'-end and a 3'-end.

[0031] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen at the 2' position of its pentose sugar compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has a modification or substitution of one or more atoms other than the 2' position, including a modification or substitution in or of a sugar, a phosphate group, or a base.

[0032] As used herein, the term "oligonucleotide" refers to a short nucleic acid, for example, a short nucleic acid less than 100 nucleotides in length. The oligonucleotide can comprise ribonucleotides, deoxyribonucleotides and / or modified nucleotides, including, for example, modified ribonucleotides. The oligonucleotide can be single-stranded or double-stranded. The oligonucleotide may or may not have a duplex region. As one group of non-limiting examples, the oligonucleotide can be, but is not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), Dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0033] As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in duplex form. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequence of the nucleotides of the covalently separated nucleic acid chains. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed between the antiparallel sequence of the nucleotides of the covalently attached nucleic acid chains. In some embodiments, the complementary base pairing of one or more duplex regions of a double-stranded oligonucleotide is formed from a single nucleic acid chain, and the single nucleic acid chain is folded (for example, via a hairpin) to provide the complementary antiparallel sequence of the nucleotides of base pairing together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are completely duplexed from each other. However, in some embodiments, a double-stranded oligonucleotide comprises partially duplexed, for example, two covalently separated nucleic acid chains with an overhang at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises the antiparallel sequence of nucleotides, which are partially complementary, and therefore, can have one or more mispairings, and the mispairings can include internal mispairings or terminal mispairings.

[0034] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientations relative to a target RNA (i.e., a complement component C3 gene). In some embodiments of the present disclosure, double-stranded RNA (dsRNA) triggers degradation of a target RNA (e.g., mRNA) through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally, the majority of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each strand or both strands may also comprise one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Additionally, as used herein, "RNAi" may comprise ribonucleotides having chemical modifications; RNAi may comprise substantial modifications at multiple nucleotides.

[0035] As used herein, the terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent" are used interchangeably herein and refer to an agent that comprises RNA, as such terms are defined herein, and that mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates, for example, inhibits expression of complement component C3 in cells, for example, cells within an individual, such as a mammalian individual.

[0036] As used herein, "conjugation" refers to the covalent attachment of two or more chemical moieties, each with a specific function, to each other; accordingly, "conjugate" refers to a compound formed by covalent attachment of the chemical moieties. Furthermore, "siRNA conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties with a specific function to siRNA. Hereinafter, the siRNA conjugates of the present disclosure will sometimes be referred to as "conjugates." siRNA conjugates should be understood as a general term for siRNA conjugates, the first siRNA conjugate or the second siRNA conjugate, or the siRNA sense strand conjugate or the siRNA antisense strand conjugate, depending on the context.

[0037] As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term "modified nucleotide" encompasses substitutions, additions, or removals of internucleoside linkages, sugar moieties, or nucleobases, such as functional groups or atoms. Modifications suitable for use with the agents of the present disclosure include all types of modifications disclosed herein or known in the art.

[0038] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded RNAi. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0039] As used herein, the term "naked sequence" refers to an unmodified nucleotide sequence.

[0040] As used herein, the term "inhibit" is used interchangeably with "knockdown," "reduction," "silencing," "downregulate," "suppression," and other similar terms, and includes any degree of inhibition.

[0041] The phrase "inhibiting the expression of complement component C3" is intended to refer to inhibiting the expression of any complement component C3 gene (such as, for example, a mouse complement component C3 gene, a rat complement component C3 gene, a monkey complement component C3 gene, or a human complement component C3 gene), as well as variants or mutants of complement component C3 genes. Thus, in the context of genetically manipulated cells, cell populations, or organisms, the complement component C3 gene can be a wild-type complement component C3 gene, a mutant complement component C3 gene, or a transgenic complement component C3 gene.

[0042] "Inhibiting complement component C3 gene expression" includes inhibition of complement component C3 gene expression at any level, for example, at least partial inhibition of complement component C3 gene expression. Complement component C3 gene expression can be assessed based on the level or change in the level of any variable associated with complement component C3 gene expression, for example, complement component mRNA levels or complement component C3 protein levels, or indirectly reflects inhibition of C3 protein levels by inhibiting the mRNA levels of Gluc and C3 fusion protein genes, thereby inhibiting the level of Gluc protein.

[0043] Levels can be assessed in individual cells or cell populations, including, for example, a sample from a subject. It is understood that complement component C3 is primarily expressed in the liver, but is also expressed in the brain, gall bladder, heart, and kidneys, and is present in the circulation.

[0044] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with complement component C3 expression compared to a control level. The control level can be any type of control level used in the art, for example, a pre-dose baseline level, or a level determined from a similar subject that has not been treated or that has been treated with a control, such as, for example, a buffer-only control or an inactive agent control.

[0045] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and characteristics of free alkali or free acid, which are not biologically or otherwise undesirable. These salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (particularly hydrochloric acid) and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methylsulfonic acid, ethylsulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine. In addition, these salts can be prepared by adding inorganic bases or organic bases to the free acid. Salts derived from inorganic bases include but are not limited to alkali metal salts (such as sodium salts, potassium salts and lithium salts), ammonium salts, alkaline earth metal salts (such as calcium salts and magnesium salts). Salts derived from organic bases include, but are not limited to, salts formed with the following organic bases (e.g., organic amines): primary amines, secondary amines, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resins. The oligonucleotides of the present disclosure may also exist in the form of zwitterions. Particularly preferred pharmaceutically acceptable salts of the present disclosure are sodium salts, lithium salts, potassium salts, and trialkylammonium salts.

[0046] As used herein, the term "subject" refers to an animal that expresses the target gene endogenously or heterologously, such as a mammal, including primates (such as humans, non-human primates, such as monkeys and chimpanzees), non-primates (such as cows, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats or mice) or birds. In one embodiment, the subject is a human.

[0047] As used herein, the term "treating" or "treatment" refers to a beneficial or desired result, such as reducing at least one sign or symptom of a complement component C3-associated disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesirable complement component C3 expression; reducing the extent of undesirable complement component C3 activation or stabilization; ameliorating or alleviating undesirable complement component C3 activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesirable complement component C3 expression. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment.

[0048] As used herein, the terms "prevention" or "preventing" when used in reference to a disease or condition that would benefit from a reduction in complement component C3 gene expression or complement component C3 protein production.

[0049] As used herein, the term "therapeutically effective amount" is intended to encompass an amount of an RNAi agent that, when administered to a subject suffering from a complement component C3-associated disorder, is sufficient to affect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more disease symptoms). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of previous or concomitant treatment (if any), and other individual characteristics of the subject to be treated.

[0050] As used herein, the term "prophylactically effective amount" is intended to encompass an amount of an RNAi agent that, when administered to a subject suffering from a complement component C3-associated disorder, is sufficient to prevent or ameliorate the disorder or one or more symptoms of the disorder. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of the disease that develops later. A "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of disease risk, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concomitant therapy (if any), and other individual characteristics of the patient being treated.

[0051] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting the expression of complement component C3, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having a sequence having at least 80% sequence identity to a sequence shown in any one of SEQ ID NOs. 1-49 and 51-305, or a fragment thereof, or a modified sequence thereof, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity; the antisense strand having a sequence having at least 80% sequence identity to a sequence shown in any one of SEQ ID NOs. 306-586, 588-586, and 1002-1009, or a fragment thereof, or a modified sequence thereof, preferably a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0052] In some embodiments of the present disclosure, wherein each strand is independently 19 to 25 nucleotides in length.

[0053] In some embodiments of the present disclosure, the antisense strand is 19 to 23 nucleotides in length.

[0054] In some embodiments of the present disclosure, the sense strand is 19 to 23 nucleotides in length.

[0055] In some embodiments of the present disclosure, the oligonucleotide comprises a 5' and / or 3'-overhang sequence having a length of one or more nucleotides, wherein the 5' and / or 3'-overhang sequence is present on the antisense strand and / or the sense strand. In one embodiment, the antisense strand of the oligonucleotide has 1 to 10 nucleotides at the 3' end or the 5' overhang at the end. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides at the 3' end or the 5' overhang at the end. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In another embodiment, the one or more nucleotides in the overhang are replaced by nucleoside thiophosphates.

[0056] In some embodiments of the present disclosure, the antisense strand has one or two overhangs.

[0057] In some embodiments of the present disclosure, the sense strand has one or two overhangs.

[0058] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence that is 1 or 2 nucleotides in length.

[0059] In some embodiments of the present disclosure, the oligonucleotide comprises a 5'-overhang sequence that is 1 or 2 nucleotides in length.

[0060] In some embodiments of the present disclosure, the 3'-overhang sequence is present on the antisense strand. In some embodiments, the overhang sequence is selected from the group consisting of: AA, AC, AG, AU, CA, CC, CU, GA, GC, GG, GU, UA, UC, UG, UU.

[0061] In some embodiments of the present disclosure, the oligonucleotide comprises an antisense strand and a sense strand each ranging from 19 to 23 nucleotides in length.

[0062] In some embodiments of the present disclosure, the sense strand and the antisense strand form a duplex region.

[0063] In some embodiments of the present disclosure, the sense strand and the antisense strand are respectively in a 19 / 21 paired, 21 / 21 paired, 21 / 23 paired or 23 / 23 paired duplex structure.

[0064] In some embodiments of the present disclosure, the oligonucleotide comprises a 5' overhang of 1 nucleotide in length and a 3'-overhang sequence, wherein the 5' overhang and the 3'-overhang sequence are present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

[0065] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence that is 2 nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 19 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 19 nucleotides in length.

[0066] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence that is 2 nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 21 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 19 nucleotides in length.

[0067] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence that is 2 nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 21 nucleotides in length.

[0068] In some embodiments of the present disclosure, the oligonucleotide comprises a 3'-overhang sequence that is 2 nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand and the sense strand, and wherein the sense strand is 23 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex that is 21 nucleotides in length.

[0069] In some embodiments of the present disclosure, the pharmaceutically acceptable salt of the oligonucleotide can be prepared by adding an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include but are not limited to alkali metal salts (such as sodium salts, potassium salts and lithium salts), ammonium salts, alkaline earth metal salts (such as calcium salts and magnesium salts). Salts derived from organic bases (such as organic amines) include but are not limited to salts formed with the following organic bases: primary amines, secondary amines and tertiary amines, substituted amines include naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins.

[0070] In some embodiments of the present disclosure, examples of pharmaceutically acceptable salts of oligonucleotides include, but are not limited to, ammonium salts, such as salts of tertiary alkylamine compounds (e.g., triethylamine salts), metal salts such as sodium salts, potassium salts, and magnesium salts.

[0071] In some embodiments of the present disclosure, the oligonucleotide or a salt thereof may be in the form of a hydrate or a solvate.

[0072] In some embodiments of the present disclosure, the oligonucleotide comprises at least one modified nucleotide.

[0073] In some embodiments of the present disclosure, the oligonucleotide comprises at least one 2'-modified nucleotide.

[0074] In some embodiments of the present disclosure, the 2'-modified nucleotides are selected from one or more of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-fluoro modified nucleotides, and 2'-deoxy nucleotides.

[0075] In some embodiments of the present disclosure, the 2'-modification is a modification selected from the group consisting of 2'-methoxy, 2'-acetamido, 2'-aminoethyl, 2'-fluoro, 2'-O-methoxyethyl.

[0076] In some embodiments of the present disclosure, the oligonucleotide has a 5'-phosphate analog modified nucleotide at the 5' end; preferably, the 5'-phosphate analog modified nucleotide has a vinyl phosphonate modified nucleotide as shown in formula (I), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl, and Base represents a nucleic acid base selected from A, G, C, T and U; preferably, the 5'-phosphate analog modified nucleotide has a vinyl phosphate modified nucleotide as shown in formula (II), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl; more preferably, the 5'-phosphonate analog modified nucleotide is APU as shown in formula (III) or VPUm as shown in formula (IV);

[0077] In some embodiments of the present disclosure, the oligonucleotide comprises a 6-(3-(2-carboxyethyl)phenyl)purine modified nucleotide; preferably, the oligonucleotide comprises formula M, which is a 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide shown in formula (V);

[0078] In some embodiments of the present disclosure, the oligonucleotide comprises a uridine-2'-phosphate (U-2'5') selected from the group consisting of uridine-2'-phosphate (U-2'5') of formula (VI), guanosine-2'-phosphate (G-2'5') of formula (VII); cytidine-2'-phosphate (C-2'5') of formula (VIII); adenosine-2'-phosphate (A-2'5') of formula (IX); and thymidine-2'-phosphate (T-2'5') of formula (X);

[0079] In some embodiments of the present disclosure, the oligonucleotide comprises at least one modified internucleotide linkage.

[0080] In some embodiments of the present disclosure, at least one modified internucleotide bond is a phosphorothioate bond. The phosphorothioate internucleotide bond modification can occur on any nucleotide of the sense strand, antisense strand, or both strands at any position in the strand. For example, the internucleotide bond modification can occur on each nucleotide on the sense strand or antisense strand; each internucleotide bond modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand can contain two internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the internucleotide bond modification on the sense strand can have an offset relative to the alternating pattern of the internucleotide bond on the antisense strand. In one embodiment, the double-stranded RNAi agent includes 4 to 8 phosphorothioate internucleotide bonds. In some embodiments, the antisense strand includes two phosphorothioate internucleotide bonds at the 5' end and two phosphorothioate internucleotide bonds at the 3' end, and the sense strand includes at least two phosphorothioate internucleotide bonds at the 5' end or the 3' end.

[0081] In some embodiments of the present disclosure, the sense strand is selected from the unmodified oligonucleotide of any one of SEQ ID NO. 2, 5, 6, 15, 18, 20, 37, 41, 48, 67, 81, 91, 115, 117, 119, 125, 133, 163, 175, 183, 189, 208, 213, 215, 216, 217, 224, 225, 227, 229, 230, 235, 237, 244, 247, 250, 251, 252, 253, 255, 256, 257, 264, 267, 271, 277, 278, 288, 289, 291, 302, 303, or the modified oligonucleotide of any one of SEQ ID NO. 588-791; the antisense strand is selected from the unmodified oligonucleotide of any one of SEQ ID NO. 307, 310, 311, 320, 323, 325, 342, 346, 353, 372, 386, 396, 420, 422, 424, 430, 438, 468, 480, 488, 494, 513, 518, 520, 521, 522, 529, 530, 532, 534, 535, 540, 542, 549, 552, 555, 556, 557, 558, 560, 561, 562, 569, 572, 573, 574, 575, 576, 578, 579, 580, 581, 582, 584, 585, 1009, or an unmodified oligonucleotide of any one of SEQ ID The modified oligonucleotide according to any one of NOs. 793-1001 and 1010-1021.

[0082] In some embodiments of the present disclosure, the oligonucleotide comprises any one selected from the following sense and antisense strand combinations:

[0083] (1) the sense strand comprises the sequence shown in SEQ ID NO. 2, and the antisense strand comprises the sequence shown in SEQ ID NO. 307;

[0084] (2) the sense strand comprises the sequence shown in SEQ ID NO. 5, and the antisense strand comprises the sequence shown in SEQ ID NO. 310;

[0085] (3) the sense strand comprises the sequence shown in SEQ ID NO. 6, and the antisense strand comprises the sequence shown in SEQ ID NO. 311;

[0086] (4) the sense strand comprises the sequence shown in SEQ ID NO. 15, and the antisense strand comprises the sequence shown in SEQ ID NO. 320;

[0087] (5) the sense strand comprises the sequence shown in SEQ ID NO. 18, and the antisense strand comprises the sequence shown in SEQ ID NO. 323;

[0088] (6) the sense strand comprises the sequence shown in SEQ ID NO. 37, and the antisense strand comprises the sequence shown in SEQ ID NO. 342;

[0089] (7) the sense strand comprises the sequence shown in SEQ ID NO. 41, and the antisense strand comprises the sequence shown in SEQ ID NO. 346;

[0090] (8) the sense strand comprises the sequence shown in SEQ ID NO. 48, and the antisense strand comprises the sequence shown in SEQ ID NO. 353;

[0091] (9) the sense strand comprises the sequence shown in SEQ ID NO. 67, and the antisense strand comprises the sequence shown in SEQ ID NO. 372;

[0092] (10) the sense strand comprises the sequence shown in SEQ ID NO. 91, and the antisense strand comprises the sequence shown in SEQ ID NO. 396;

[0093] (11) the sense strand comprises the sequence shown in SEQ ID NO. 117, and the antisense strand comprises the sequence shown in SEQ ID NO. 422;

[0094] (12) the sense strand comprises the sequence shown in SEQ ID NO. 81, and the antisense strand comprises the sequence shown in SEQ ID NO. 386;

[0095] (13) the sense strand comprises the sequence shown in SEQ ID NO. 189, and the antisense strand comprises the sequence shown in SEQ ID NO. 494;

[0096] (14) the sense strand comprises the sequence shown in SEQ ID NO. 213, and the antisense strand comprises the sequence shown in SEQ ID NO. 518;

[0097] (15) the sense strand comprises the sequence shown in SEQ ID NO. 217, and the antisense strand comprises the sequence shown in SEQ ID NO. 522;

[0098] (16) the sense strand comprises the sequence shown in SEQ ID NO. 225, and the antisense strand comprises the sequence shown in SEQ ID NO. 530;

[0099] (17) the sense strand comprises the sequence shown in SEQ ID NO. 230, and the antisense strand comprises the sequence shown in SEQ ID NO. 535;

[0100] (18) the sense strand comprises the sequence shown in SEQ ID NO. 237, and the antisense strand comprises the sequence shown in SEQ ID NO. 542;

[0101] (19) the sense strand comprises the sequence shown in SEQ ID NO. 247, and the antisense strand comprises the sequence shown in SEQ ID NO. 552;

[0102] (20) the sense strand comprises the sequence shown in SEQ ID NO. 267, and the antisense strand comprises the sequence shown in SEQ ID NO. 572;

[0103] (21) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 572;

[0104] (22) the sense strand comprises the sequence shown in SEQ ID NO. 277, and the antisense strand comprises the sequence shown in SEQ ID NO. 573;

[0105] (23) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 574;

[0106] (24) the sense strand comprises the sequence shown in SEQ ID NO. 288, and the antisense strand comprises the sequence shown in SEQ ID NO. 575;

[0107] (25) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 576;

[0108] (26) the sense strand comprises the sequence shown in SEQ ID NO. 115, and the antisense strand comprises the sequence shown in SEQ ID NO. 420;

[0109] (27) the sense strand comprises the sequence shown in SEQ ID NO. 291, and the antisense strand comprises the sequence shown in SEQ ID NO. 578;

[0110] (28) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 579;

[0111] (29) the sense strand comprises the sequence shown in SEQ ID NO. 302, and the antisense strand comprises the sequence shown in SEQ ID NO. 584;

[0112] (30) the sense strand comprises the sequence shown in SEQ ID NO. 303, and the antisense strand comprises the sequence shown in SEQ ID NO. 585;

[0113] (31) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 582;

[0114] (32) the sense strand comprises the sequence shown in SEQ ID NO. 277, and the antisense strand comprises the sequence shown in SEQ ID NO. 580;

[0115] (33) the sense strand comprises the sequence shown in SEQ ID NO. 288, and the antisense strand comprises the sequence shown in SEQ ID NO. 581;

[0116] (34) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 1005;

[0117] (35) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 1009;

[0118] wherein each strand is independently 19 to 25 nucleotides in length.

[0119] In some embodiments of the present disclosure, the oligonucleotide comprises any one selected from the following sense and antisense strand combinations:

[0120] (1) the sense strand comprises the sequence shown in SEQ ID NO. 2, and the antisense strand comprises the sequence shown in SEQ ID NO. 307;

[0121] (2) the sense strand comprises the sequence shown in SEQ ID NO. 18, and the antisense strand comprises the sequence shown in SEQ ID NO. 323;

[0122] (3) the sense strand comprises the sequence shown in SEQ ID NO. 48, and the antisense strand comprises the sequence shown in SEQ ID NO. 353;

[0123] (4) the sense strand comprises the sequence shown in SEQ ID NO. 115, and the antisense strand comprises the sequence shown in SEQ ID NO. 420;

[0124] (5) the sense strand comprises the sequence shown in SEQ ID NO. 117, and the antisense strand comprises the sequence shown in SEQ ID NO. 422;

[0125] (6) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 574;

[0126] (7) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 579;

[0127] (8) the sense strand comprises the sequence shown in SEQ ID NO. 302, and the antisense strand comprises the sequence shown in SEQ ID NO. 584;

[0128] (9) the sense strand comprises the sequence shown in SEQ ID NO. 303, and the antisense strand comprises the sequence shown in SEQ ID NO. 585;

[0129] (10) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 582;

[0130] (11) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 572;

[0131] (12) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 576;

[0132] (13) The sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 1009.

[0133] In some embodiments of the present disclosure, the oligonucleotide comprises any one selected from the following sense and antisense strand combinations:

[0134] (1) the sense strand comprises the sequence shown in SEQ ID NO. 588, and the antisense strand comprises the sequence shown in SEQ ID NO. 793;

[0135] (2) the sense strand comprises the sequence shown in SEQ ID NO. 589, and the antisense strand comprises the sequence shown in SEQ ID NO. 794;

[0136] (3) the sense strand comprises the sequence shown in SEQ ID NO. 590, and the antisense strand comprises the sequence shown in SEQ ID NO. 795;

[0137] (4) the sense strand comprises the sequence shown in SEQ ID NO. 591, and the antisense strand comprises the sequence shown in SEQ ID NO. 796;

[0138] (5) the sense strand comprises the sequence shown in SEQ ID NO. 592, and the antisense strand comprises the sequence shown in SEQ ID NO. 797;

[0139] (6) the sense strand comprises the sequence shown in SEQ ID NO. 593, and the antisense strand comprises the sequence shown in SEQ ID NO. 798;

[0140] (7) the sense strand comprises the sequence shown in SEQ ID NO. 594, and the antisense strand comprises the sequence shown in SEQ ID NO. 799;

[0141] (8) the sense strand comprises the sequence shown in SEQ ID NO. 595, and the antisense strand comprises the sequence shown in SEQ ID NO. 800;

[0142] (9) the sense strand comprises the sequence shown in SEQ ID NO. 596, and the antisense strand comprises the sequence shown in SEQ ID NO. 801;

[0143] (10) the sense strand comprises the sequence shown in SEQ ID NO. 597, and the antisense strand comprises the sequence shown in SEQ ID NO. 802;

[0144] (11) the sense strand comprises the sequence shown in SEQ ID NO. 599, and the antisense strand comprises the sequence shown in SEQ ID NO. 804;

[0145] (12) the sense strand comprises the sequence shown in SEQ ID NO. 601, and the antisense strand comprises the sequence shown in SEQ ID NO. 806;

[0146] (13) the sense strand comprises the sequence shown in SEQ ID NO. 634, and the antisense strand comprises the sequence shown in SEQ ID NO. 839;

[0147] (14) the sense strand comprises the sequence shown in SEQ ID NO. 674, and the antisense strand comprises the sequence shown in SEQ ID NO. 879;

[0148] (15) the sense strand comprises the sequence shown in SEQ ID NO. 698, and the antisense strand comprises the sequence shown in SEQ ID NO. 903;

[0149] (16) the sense strand comprises the sequence shown in SEQ ID NO. 702, and the antisense strand comprises the sequence shown in SEQ ID NO. 907;

[0150] (17) the sense strand comprises the sequence shown in SEQ ID NO. 710, and the antisense strand comprises the sequence shown in SEQ ID NO. 915;

[0151] (18) the sense strand comprises the sequence shown in SEQ ID NO. 715, and the antisense strand comprises the sequence shown in SEQ ID NO. 920;

[0152] (19) the sense strand comprises the sequence shown in SEQ ID NO. 722, and the antisense strand comprises the sequence shown in SEQ ID NO. 927;

[0153] (20) the sense strand comprises the sequence shown in SEQ ID NO. 732, and the antisense strand comprises the sequence shown in SEQ ID NO. 937;

[0154] (21) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 957;

[0155] (22) the sense strand comprises the sequence shown in SEQ ID NO. 756, and the antisense strand comprises the sequence shown in SEQ ID NO. 957;

[0156] (23) the sense strand comprises the sequence shown in SEQ ID NO. 762, and the antisense strand comprises the sequence shown in SEQ ID NO. 958;

[0157] (24) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 959;

[0158] (25) the sense strand comprises the sequence shown in SEQ ID NO. 773, and the antisense strand comprises the sequence shown in SEQ ID NO. 969;

[0159] (26) the sense strand comprises the sequence shown in SEQ ID NO. 774, and the antisense strand comprises the sequence shown in SEQ ID NO. 970;

[0160] (27) the sense strand comprises the sequence shown in SEQ ID NO. 776, and the antisense strand comprises the sequence shown in SEQ ID NO. 972;

[0161] (28) the sense strand comprises the sequence shown in SEQ ID NO. 776, and the antisense strand comprises the sequence shown in SEQ ID NO. 973;

[0162] (29) the sense strand comprises the sequence shown in SEQ ID NO. 595, and the antisense strand comprises the sequence shown in SEQ ID NO. 974;

[0163] (30) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 979;

[0164] (31) the sense strand comprises the sequence shown in SEQ ID NO. 756, and the antisense strand comprises the sequence shown in SEQ ID NO. 987;

[0165] (32) the sense strand comprises the sequence shown in SEQ ID NO. 762, and the antisense strand comprises the sequence shown in SEQ ID NO. 989;

[0166] (33) the sense strand comprises the sequence shown in SEQ ID NO. 773, and the antisense strand comprises the sequence shown in SEQ ID NO. 991;

[0167] (34) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 987;

[0168] (35) the sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 993;

[0169] (36) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 995;

[0170] (37) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 996;

[0171] (38) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 997;

[0172] (39) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 998;

[0173] (40) the sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 999;

[0174] (41) the sense strand comprises the sequence shown in SEQ ID NO. 756, and the antisense strand comprises the sequence shown in SEQ ID NO. 996;

[0175] (42) the sense strand comprises the sequence shown in SEQ ID NO. 762, and the antisense strand comprises the sequence shown in SEQ ID NO. 1000;

[0176] (43) the sense strand comprises the sequence shown in SEQ ID NO. 773, and the antisense strand comprises the sequence shown in SEQ ID NO. 1001;

[0177] (44) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 1013;

[0178] (45) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 1017;

[0179] (46) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 1018;

[0180] (47) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 1021;

[0181] (48) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 985;

[0182] wherein each strand is independently 19 to 25 nucleotides in length.

[0183] In some embodiments of the present disclosure, the oligonucleotide comprises any one selected from the following sense and antisense strand combinations:

[0184] (1) the sense strand comprises the sequence shown in SEQ ID NO. 588, and the antisense strand comprises the sequence shown in SEQ ID NO. 793;

[0185] (2) the sense strand comprises the sequence shown in SEQ ID NO. 592, and the antisense strand comprises the sequence shown in SEQ ID NO. 797;

[0186] (3) the sense strand comprises the sequence shown in SEQ ID NO. 595, and the antisense strand comprises the sequence shown in SEQ ID NO. 800;

[0187] (4) the sense strand comprises the sequence shown in SEQ ID NO. 599, and the antisense strand comprises the sequence shown in SEQ ID NO. 804;

[0188] (5) the sense strand comprises the sequence shown in SEQ ID NO. 601, and the antisense strand comprises the sequence shown in SEQ ID NO. 806;

[0189] (6) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 995;

[0190] (7) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 996;

[0191] (8) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 997;

[0192] (9) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 998;

[0193] (10) the sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 999;

[0194] (11) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 987;

[0195] (12) The sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 993.

[0196] (13) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 1017;

[0197] (14) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 1018;

[0198] (15) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 1021;

[0199] (16) The sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 985.

[0200] The present disclosure also provides a conjugate for inhibiting the expression of complement component C3 or a pharmaceutically acceptable salt thereof, which comprises: (i) the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, and (ii) a ligand conjugated to the aforementioned oligonucleotide or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to a targeting ligand. In some embodiments, at least one nucleotide of the aforementioned oligonucleotide or a salt thereof is conjugated to a targeting ligand to form an siRNA conjugate. The aforementioned siRNA conjugate contains the aforementioned siRNA and a conjugated group connected to the siRNA. The term "oligonucleotide salt" refers to an oligonucleotide compound in the form of a salt. Oligonucleotide salts include salts of oligonucleotide conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotide salts are advantageously present in the form of solid powder.

[0201] In general, the conjugated group comprises at least one pharmaceutically acceptable targeting ligand and an optional linker, and the siRNA, linker and targeting ligand are connected in sequence. The targeting group can be a ligand conventionally used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, the targeting ligand is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of the siRNA and the conjugated group can be at the 3' end or 5' end of the siRNA sense strand or antisense strand, or can be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugated group is at the 3' end or 5' end of the siRNA sense strand. In some embodiments, the conjugation site of the siRNA and the conjugated group is at the 3' end or 5' end of the siRNA antisense strand. In some preferred embodiments, the conjugation site of the siRNA and the conjugated group is at the 3' end of the siRNA sense strand.

[0202] In some embodiments, the targeting ligand comprises an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term "galactose derivative" includes galactose and lactose derivatives having an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. Galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyrylgalactosamine. Galactose derivatives and clusters of galactose derivatives that can be used to target oligonucleotides and other molecules to the liver in vivo are known in the art. Galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes. Binding of ASGPR ligands to ASGPR(s) facilitates cell-specific targeting to hepatocytes and endocytosis of molecules into hepatocytes. ASGPR ligands can be monomeric (eg, having a single galactose derivative) or polymeric (eg, having multiple galactose derivatives). Galactose derivatives or clusters of galactose derivatives can be linked to the 3' or 5' end of the siRNA using methods known in the art.

[0203] In some embodiments, the pharmaceutically acceptable targeting ligand in the siRNA conjugate can be galactose or N-acetylgalactosamine (GalNAc), wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent refer to the formation of an siRNA conjugate by a conjugated group containing a galactose or N-acetylgalactosamine molecule as a targeting ligand, and the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1: 1, 1: 2, 1: 3, or 1: 4. In some embodiments, the pharmaceutically acceptable targeting ligand is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0204] In some embodiments of the present disclosure, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

[0205] In some embodiments of the present disclosure, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0206] In some embodiments of the present disclosure, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0207] In some embodiments of the present disclosure, the targeting ligand is L96;

[0208] In some embodiments of the disclosed methods, expression of the complement component C3 gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or to below the level of detection. In a preferred embodiment, expression of complement component C3 is inhibited by at least 70%. It should also be understood that it may be desirable to inhibit expression of complement component C3 in certain tissues (e.g., liver) without significantly inhibiting expression in other tissues (e.g., brain). In a preferred embodiment, expression levels are determined using the assay provided in Example 2 using 50 nM, 10 nM and 1 nM siRNA concentrations in appropriate species-matched cell lines.

[0209] In certain embodiments, inhibition of in vivo expression is determined by knocking down human genes in rodents expressing human genes, for example, AAV-infected mice expressing human target genes (i.e., complement component C3), for example, when administered as a single dose, for example, at the lowest point of C3 expression after subcutaneous injection at 3 mg / kg to confirm the inhibitory effect on the human gene. Such systems are useful when the nucleic acid sequences of the human gene and the model animal gene are close enough to allow human RNAi to effectively knock down the model animal gene. RNA expression in the liver is determined using the PCR method provided in Example 2.

[0210] Inhibition of complement component C3 gene expression can be represented by a decrease in the amount of mRNA expressed by a cell line (such cells can be present, for example, in a sample derived from a subject) in which the complement component C3 gene is transcribed and which is treated (e.g., by contacting one or more cells with an RNAi disclosed herein, or by administering an RNAi disclosed herein to a subject in which cells are or were present) such that expression of the complement component C3 gene is inhibited compared to a substantially identical cell line but untreated (control cells not treated with RNAi or not treated with RNAi targeting the target gene). In a preferred embodiment, inhibition is assessed using a 10 nM siRNA concentration in a species-matched cell line using the method provided in Example 2, and is represented by 2^-ΔΔCT of the mRNA expression level in the treated cells relative to the mRNA level in the control cells using the following formula: ΔCT=CT C3 -CT GAPDH △△CT=△CT处理细胞 -△CT 对照细胞 mRNA level = 2^-△△CT

[0211] In other embodiments, inhibition of complement component C3 gene expression can be assessed based on a reduction in a parameter functionally associated with complement component C3 gene expression, e.g., complement component C3 protein levels in blood or serum from a subject. Complement component C3 gene silencing can be determined in any cell expressing complement component C3, whether endogenous or heterologous from an expression construct, and by any assay known in the art.

[0212] Inhibition of complement component C3 protein expression can be reflected by a decrease in the level of complement component C3 protein or secreted luciferase expressed by a cell or cell population or in a subject sample (e.g., the level of protein in a blood sample from a subject). As described above, to assess mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can be similarly expressed as a percentage of the protein level in a control cell or cell population, or as a change in protein level in a subject sample (e.g., blood or serum from the subject), inhibition is assessed by the method provided in Example 3 or Example 4, using the following formula, represented by the percentage of C3 expression or Gluc expression in the treated sample (e.g., blood or serum from the subject) relative to the C3 expression or Gluc expression in the control cell. Percent inhibition of mRNA = (Protein expression) 处理细胞 -Protein expression 对照细胞 ) / protein expression 对照细胞 *100%

[0213] Control cells, cell populations, or subject samples that can be used to assess inhibition of complement component C3 gene expression include cells, cell populations, or subject samples that have not been contacted with the RNAi agents of the present disclosure. For example, control cells, cell lines, or subject samples can be derived from individual subjects (e.g., human or animal subjects) prior to treatment of the subject or an appropriately matched group control with an RNAi agent.

[0214] In some embodiments of the disclosed methods, RNAi is administered to a subject so that the RNAi is delivered to a specific site within the subject. The inhibition of complement component C3 expression can be assessed by measuring the level or change of complement component C3 mRNA or complement component C3 protein or fusion secretory luciferase in a sample of fluid or tissue from a subject's specific site (e.g., liver or blood).

[0215] The present disclosure also provides methods of using the RNAi of the present disclosure or compositions comprising the RNAi of the present disclosure to inhibit the expression of complement component C3, thereby preventing or treating complement component C3-related disorders, e.g., cold agglutinin disease (CAD), warm autoimmune hemolytic anemia, and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), bullous pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

[0216] Cells suitable for treatment using the methods of the present disclosure can be any cells that express the complement component C3 gene, 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 disclosure can be mammalian cells, for example, primate cells (such as human cells, including human cells in chimeric non-human animals, or non-human primate cells, for example, monkey cells or chimpanzee cells) or non-primate cells. In certain embodiments, the cells are human cells, for example, human liver cells. In the methods of the present disclosure, the expression of complement component C3 in the cells is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or is below the detection level of the assay.

[0217] The in vivo method of the present disclosure can include administering to the subject a composition comprising RNAi, wherein the RNAi comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the complement component C3 gene of the mammal administered with the RNAi agent. The composition can be administered by any means known in the art, including but not limited to oral, intraperitoneal or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal cavity, rectal and topical (including oral and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous administration. In certain embodiments, the composition is administered by intramuscular injection.

[0218] In one aspect, the present disclosure also provides a method for inhibiting the expression of complement component C3 gene in mammals. The method comprises administering an oligonucleotide or a pharmaceutically acceptable salt thereof, a conjugate thereof, a salt thereof, or a composition thereof to a mammal. The oligonucleotide is a double-stranded RNA (dsRNA) that targets the complement component C3 gene in mammalian cells and maintains the mammal for a sufficient time to obtain degradation of the mRNA transcript of the complement component C3 gene, thereby inhibiting the expression of the complement component C3 protein in the cell. The reduction in gene expression can be by any method known in the art and by methods, for example, qRT-PCR as described herein, for example, as assessed in Example 2. The reduction in protein product can be assessed by any method known in the art (e.g., ELISA). In other embodiments, a blood sample is used as a subject sample to monitor the reduction in complement component C3 protein expression.

[0219] The present disclosure also provides methods of treating in a subject in need thereof, e.g., a subject diagnosed with a complement component C3-associated disorder, such as cold agglutinin disease (CAD), warm autoimmune hemolytic anemia, and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), bullous pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), or C3 glomerulopathy.

[0220] In one embodiment, the complement component C3 related disease is cold agglutinin disease (CAD). CAD is an autoimmune complement component C3-induced hemolytic anemia, in which cold exposure can cause clinical symptoms (e.g., livedo reticularis or acrocyanosis) and hemolytic anemia associated with red blood cell (RBC) agglutination in cold parts of the body. Cold agglutinins are IgM antibodies that can recognize antigens on red blood cells (RBC) at temperatures below normal core body temperature. They can cause RBC agglutination, complement activation, and extravascular hemolysis, leading to anemia, usually without hemoglobinuria. CAD can be primary CAD (also known as idiopathic CAD) or secondary CAD. In subjects suffering from primary CAD, cold agglutinins can cause RBC agglutination in the absence of underlying disease. And extravascular hemolysis. In subjects with secondary CAD (also known as cold agglutinin syndrome, or CAS), cold agglutinins appear in the context of an underlying disease, such as viral interference, autoimmune disease, or lymphoid malignancy (see, e.g., Berentsen (2015) Transfus Med Hemother 42:303-310).

[0221] In one embodiment, the complement component C3-related disease is warm autoimmune hemolytic anemia. Warm autoimmune hemolytic anemia is an autoimmune complement component C3-induced hemolytic anemia in which red blood cells (RBCs) agglutinate at body sites at temperatures equal to or higher than normal body temperature and the complement system is activated due to IgG antibodies directed against blood group antigens, resulting in hemolytic anemia. Warm autoimmune hemolytic anemia is the most common type of autoimmune hemolytic anemia, accounting for approximately 70% to 80% of all adult cases and approximately 50% of pediatric cases. Approximately half of cases of warm autoimmune hemolytic anemia are primary, because no specific cause can be found, while the remainder are thought to be secondary to lymphoproliferative syndromes; malignant disorders, including chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, and solid tumors; rheumatic diseases, particularly systemic lupus erythematosus; infections (primarily viral); drugs; frequent use of cephalosporins and piperacillin; or previous blood transfusion or transplantation (see, e.g., Berentsen (2015) Transfus Med Hemother 42:303-310).

[0222] In one embodiment, the complement component C3-related disease is paroxysmal nocturnal hemoglobinuria (PNH). PNH can be classic PNH or PNH in the context of another bone marrow failure syndrome and / or myelodysplastic syndrome (MDS), e.g., cytopenia. PNH is an acquired autoimmune disease that can lead to premature death and impaired hematopoiesis, characterized by complement-mediated hemolytic anemia, thrombotic tendency, and bone marrow failure (see, e.g., Risitano (2013) Adv Exp Med Biol 735:155).

[0223] In one embodiment, the complement component C3-associated disease is lupus nephritis (LN), any of class I to class VI lupus nephritis. LN is a glomerulonephritis caused by systemic lupus erythematosus (SLE). Lupus nephritis occurs due to the deposition of immune complexes in any or all renal compartments, including the glomeruli, tubules, and interstitium. IgG is the most common antibody, but IgM and IgA can also be seen. These autoantibodies lead to activation of the classical and alternative complement pathways, and therefore C1, C3, and properdin can be found in biopsies.

[0224] In one embodiment, the complement component C3-associated disease is bullous pemphigoid. Bullous pemphigoid is an autoimmune blistering disease caused by autoantibodies against type XVII collagen (COL17), which activates complement and subsequently recruits inflammatory cells at the dermal / epidermal junction. Bullous pemphigoid is the most common autoimmune blistering disorder and is characterized by tight blisters accompanied by itchy urticarial erythema and plaques throughout the body.

[0225] In one embodiment, the complement component C3 related disease is pemphigus, for example, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Pemphigus is a group of rare chronic herpes diseases characterized by IgG autoantibodies and intracellular deposition of IgG and C3c to multiple desmosomal transmembrane glycoproteins. Pemphigus vulgaris patients typically present with oral mucosal lesions, followed by skin involvement, and autoantibodies to epithelial adhesion proteins desmoglein 3 and / or desmoglein 1. In pemphigus foliaceus, the lesions are confined to the skin, do not involve mucosa, and autoantibodies are to desmoglein 1. In one embodiment, pemphigus is pemphigus vulgaris (PV). In another embodiment, pemphigus is pemphigus foliaceus (PF).

[0226] In one embodiment, the complement component C3-associated disease is C3 glomerulopathy. C3 glomerulopathy is characterized by activation of the alternative complement cascade and deposition of complement component C3 without any immunoglobulin deposition in the glomeruli of the kidney.

[0227] The RNAi disclosed herein can be administered as "free RNAi". Free RNAi is administered without a pharmaceutical composition. Naked RNAi can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, lactate, tartrate, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing RNAi can be adjusted to make it suitable for administration to a subject.

[0228] Administration of RNAi according to the methods disclosed herein can result in the prevention or treatment of complement component C3 related disorders, for example, cold agglutinin disease (CAD), warm autoimmune hemolytic anemia, and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), bullous pemphigoid, pemphigus, such as pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy. A therapeutic amount of RNAi can be administered to the subject, such as from about 0.01 mg / kg to about 200 mg / kg. Preferably, 1 mg / kg to about 50 mg / kg. RNAi is preferably administered subcutaneously, that is, by subcutaneous injection. One or more injections can be used to deliver the desired dose of RNAi to the subject. The injection can be repeated over a period of time.

[0229] In some embodiments, RNAi is administered at a fixed dose of about 10 mg to about 800 mg. In some embodiments, RNAi is administered to a subject at a fixed dose of about 10 to 50 mg, about 50 mg to about 200 mg, about 200 mg to about 400 mg, or about 400 mg to about 800 mg. In some embodiments, RNAi is administered to a subject at a fixed dose of about 10 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, 500 mg, about 600 mg, about 700 mg, or about 800 mg.

[0230] Can be regularly repeated administration. In certain embodiments, after the initial treatment regimen, treatment can be carried out at a lower frequency. Repeated dosing regimens may include regular administration of a therapeutic amount of RNAi, such as once a month to once a year. In certain embodiments, RNAi is administered from about once a month to about once every three months, or from about once every three months to about once every six months, or even once a year.

[0231] The present disclosure further provides for the combination of RNAi agents or pharmaceutical compositions thereof with other drugs and / or other treatments (e.g., known drugs and / or known treatments, such as, for example, those currently used to treat these conditions) for the treatment of subjects who would benefit from the reduction and / or inhibition of C3 gene expression, e.g., subjects with a C3-related disease. For example, other therapeutic agents and treatments suitable for treating subjects who would benefit from the reduction of C3 expression (e.g., subjects with a complement component C3-related disease) include plasma electrophoresis, thrombolytic therapy (e.g., streptokinase), antiplatelet drugs, folic acid, corticosteroids; immunosuppressants; antithrombotic agents, complement inhibitors, adrenergic drugs, drugs that interfere with proinflammatory cytokine signaling (e.g., TNF-α or IL-1) (e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors); C3 cyclic peptide inhibitors; other therapeutic agents also include anti-complement component C5 antibodies or antigen-binding fragments thereof (e.g., eculizumab).

[0232] Example

[0233] Example 1. Preparation of targeting ligands and siRNA

[0234] Unless the source of a reagent is specifically given herein, such reagent can be obtained from any molecular biology reagent supplier at quality / purity standards appropriate for molecular biology.

[0235] Abbreviation for nucleotide monomers used in nucleic acid sequence representation.

[0236] Table A. Nucleotide monomer abbreviations used in nucleic acid sequence representation

[0237] Preparation of targeting ligands

[0238] The preparation of L96 was carried out according to the method described in patent CN104717982B.

[0239] Preparation of oligonucleotides

[0240] (1) Preparation of siRNA

[0241] First, a computer-based algorithm was used to generate candidate oligonucleotide sequences complementary to human C3 mRNA (NM_000064.3, Table 1). Some of these sequences were also complementary to or had no more than two mismatches with cynomolgus macaque C3 mRNA (XM_005587719.3, Table 1). Some of these sequences were designed as double-stranded siRNAs with a 19 / 21 sense / antisense pairing, with the antisense strand possessing two overhangs complementary to the mRNA sequence; in some cases, the antisense strand had non-complementary UU overhangs. Other sequences were designed as double-stranded siRNAs with a 21 / 23 sense / antisense pairing, with the antisense strand possessing two overhangs complementary to the mRNA sequence. Other sequences were designed as double-stranded siRNAs with 21 / 21 or 23 / 23 pairings. In some of these complementary pairs, the first base at the 5' end of the antisense strand (the last base at the 3' end of the sense strand) was replaced with a base that did not match the C3 mRNA sequence.

[0242] Table 1 Human and cynomolgus macaque C3 mRNA sequences

[0243] The siRNA sequence is synthesized separately on a solid support via a sense strand (SS) and an antisense strand (AS), and is obtained after deprotection, cleavage, purification, annealing, purification, and lyophilization.

[0244] Solid-phase synthesis (Figure 2): Sense and antisense strands are synthesized separately on a solid support using phosphoramidite technology using an automated oligonucleotide synthesizer. Examples of such synthesizers include the AKTA Oligopilot (Cytiva) and the Dr. Oligo 192XLc (Kunshan Berleke Precision Instrument Co., Ltd.). Solid-phase synthesis begins at the 3' end of the sequence and sequentially couples monomers into the sequence. Each coupling of a phosphoramidite monomer involves four chemical steps: 1) unblocking or deprotection (removal of the hydroxyl protecting group); 2) coupling; 3) oxidation; and 4) capping. All phosphoramidite monomers, reagents, and purification consumables used were commercially available, including various phosphoramidite monomers (e.g., 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (e.g., 40 wt% aqueous methylamine solution, 28 wt% aqueous ammonium hydroxide solution) purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used herein are described in US20130178612A1 and US2015100197A1, among others; the synthesis methods for sequences containing VPUm and APU structures are described in J. Med. Chem. 2018, 61, 734-744.

[0245] (2) Preparation of double-stranded RNA reagent

[0246] (a) Synthesis of the positive chain

[0247] The solid-phase phosphoramidite method is a well-established method for oligonucleotide synthesis. It utilizes a computer-controlled synthesizer and is carried out within a stainless steel synthesis column. Sense strand synthesis begins with a solid support loaded with a targeting ligand (e.g., L96) or directly on the solid support. The solid-phase synthesizer controls different pipelines, injecting various raw materials, reagents, and solvents in a 3' to 5' sequence order, ligating phosphoramidite nucleoside monomers one by one. The reaction process involves four cycles: DMT protection group removal, condensation, oxidation or thiolation, and end-capping. Each cycle adds a single nucleotide unit to the oligonucleotide, yielding sequences of 19 or 21 nucleotide units. Following synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and the synthesized sequence is cleaved from the solid support via aminolysis. The resulting product is filtered, the filter cake washed with ethanol, and the filtrate and washings are collected and concentrated to yield the crude sense strand. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to yield the desired sense strand. Among them, in the synthesizer, the siRNA sense chain conjugate is synthesized starting from the solid support loaded with the targeting ligand (such as L96); and the siRNA is synthesized directly starting from the solid support.

[0248] (b) Synthesis of antisense strand

[0249] The synthesis of the antisense strand is similar to that of the sense strand. A solid-phase synthesizer controls different pipelines to inject different raw materials, reagents, and solvents in the order of 3' to 5' of the sequence, connecting the phosphoramidite nucleoside monomers one by one. The reaction process includes four cycles: DMT protection group removal, condensation reaction, oxidation or thiolation reaction, and end-capping reaction. Each cycle connects a nucleotide unit to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After synthesis, the protecting group (2-cyanoethyl) is removed on a solid-phase synthesis column, and the synthesized sequence is cleaved from the solid phase support by aminolysis. The residue is filtered, the filter cake is washed with ethanol, and the filtrate and washing liquid are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltration, and lyophilization to obtain the target antisense strand siRNA.

[0250] (c) Preparation of double-stranded siRNA

[0251] Dissolve the AS and SS strands separately in injection water, mix at a defined ratio (1.01:1.0-1.2:1.0), incubate at 30-50°C for 30-90 minutes, cool to room temperature, and freeze-dry to obtain double-stranded siRNA.

[0252] According to the same method, the double-stranded siRNA agents shown in Tables 2, 3 and 4 below were prepared.

[0253] In Tables 2, 3, and 4, "G," "C," "A," "U," "T," and "I" generally represent nucleotides based on guanine, cytosine, adenine, uracil, thymine, and hypoxanthine, respectively. The naked sequences in Tables 2, 3, and 4 refer to unmodified oligonucleotide sequences.

[0254] Modifications: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents phosphorothioate; VPUm is 2'-methoxy-modified uridine; M formula is 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide; L96 is N-[tris(GalNAc-alkyl)amidodecanoyl]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3); dA represents 2'-deoxyadenosine-3'-phosphate; dG represents 2'-deoxyguanosine-3'-phosphate; dC represents 2'-deoxycytidine-3'-phosphate; dU represents 2'-deoxyuridine-3'-phosphate.

[0255] VPUm:

[0256] M:

[0257] L96:

[0258] Table 2 Oligonucleotide naked sequences

[0259] Table 3 Oligonucleotide modified sequences

[0260] Table 4 siRNA sequences with targeting ligands

[0261] Example 2. In vitro activity screening of naked C3-siRNA sequences

[0262] (1) Cell culture and transfection:

[0263] Human hepatocellular carcinoma cells (undifferentiated) (also known as HLE cells) (Wuhan Punosai Life Science Technology Co., Ltd., Catalog No. CL-0651) and Hep3B cells (Wuhan Punosai Life Science Technology Co., Ltd., Catalog No. CL-0102) were cultured in a 37°C, 5% CO2 incubator using DMEM medium (Synopharm (Shanghai) Biotechnology Co., Ltd., Catalog No. iCell-0001) supplemented with 10% FBS (GIBCO, 12483020) and 1% penicillin-streptomycin (GIBCO, 15140-122). When the cell confluence reached 90%, they were digested with trypsin-EDTA (Thermo, 25200-072) and counted using a cell counter (Countstar, IC1000). 190 μl of cell suspension / well was inoculated into a 96-well plate. The inoculation number of HLE cells was: 5*10 4 cells / well, the seeding number of Hep3B cells is 2*10 4 cells / well and wait for them to adhere to the wall for transfection the next day.

[0264] Transfection was performed using Lipofectamine™ RNAiMAX (thermofisher, 13778150). 2.2 μl (2 μM) of the diluted compound, 19.1 μl Opti-MEM (thermofisher, 1105821), and 0.7 μl of RNAiMAX were mixed to form a transfection complex. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates for each complex), 10 μl per well, with a final siRNA concentration of 10 nM. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0265] (2) RNA extraction and detection

[0266] (i) Total RNA was extracted using RNA-Quick Purification Kit (RN001, Yishan Biotechnology):

[0267] Remove the 12-well plate from the incubator, aspirate the medium, wash once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a fresh 1.5 ml centrifuge tube. Add 500 μl of absolute ethanol to the lysed cells and mix thoroughly (if a precipitate forms, this is normal; continue with the procedure). Invert the centrifuge tube several times or pipette vigorously 10 times to disperse the precipitate. Then add the liquid to the spin column, place the tubes symmetrically in a centrifuge (Eppendorf, 5430), and centrifuge at 4000 × g for 1 minute. Remove the centrifuge tube and add 500 μl of wash buffer to the column. Centrifuge at 12000 × g for 1 minute. After centrifugation, remove the column, discard the waste liquid, and return the RNA column to the collection tube. Centrifuge the empty tube once to remove any residual wash buffer. Place the column in a clean RNase-free 1.5 ml centrifuge tube and air dry it with the lid open for 2 minutes. Add 30 μl of elution buffer to the center of the RNA column membrane. Let stand at room temperature for 2 minutes. Centrifuge at 2000 × g for 1 minute to elute the RNA, then place on ice. Measure the eluted RNA concentration for subsequent experiments. The extracted RNA can be used immediately for subsequent experiments or stored at -80°C for later use.

[0268] (ii) Use cDNA was synthesized using IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novozyme, R223-01):

[0269] In an RNase-free centrifuge tube, prepare a mixture of 4 μl of 4×g DNA wiper mix, 1 μg of template RNA, and 16 μl of RNase-free ddH₂O to remove genomic DNA. Gently pipette to mix thoroughly and incubate at 42°C for 2 minutes. Then, add 4 μl of 5× HiScript II qRT SuperMix II directly to the reaction tube and gently pipette to mix thoroughly. Place the tube in a PCR instrument (Applied Biosystems, 9700) and cycle at 50°C for 15 minutes, 85°C for 5 seconds, and hold at 4°C. The product can be used immediately for qPCR reactions or stored at -20°C and used within six months. For long-term storage, aliquot and store at -80°C. Avoid repeated freeze-thaw cycles for cDNA.

[0270] (iii) qPCR quantification using ChamQ SYBR qPCR Master Mix (Novozymes, Q311-02):

[0271] A mixture of 10 μl 2× ChamQ SYBR qPCR Master Mix, 0.5 μl Forword primer (Ruiboxing), 0.5 μl Reverse primer (Ruiboxing), 1 μl Template cDNA, and 8 μl ddH2O was prepared to 20 μl system. Each sample was replicated three times. The 96-well plate was placed in a qPCR instrument (ROCGENE, Archimed) and the following program was performed: initial denaturation, 95°C, 30 sec; amplification, 95°C, 10 sec, 60°C, 30 sec, 40 cycles; melting curve, 95°C, 15 sec, 60°C, 60 sec, 95°C, 15 sec.

[0272] (3) Data statistical analysis:

[0273] Export the data to EXCEL format and use CT C3 -CT GAPDH The control group was normalized. In order to calculate the fold change of relative silencing efficiency, the data were analyzed using the △△CT method, and the mean and standard deviation of the three parallel repeated data were calculated.

[0274] The results of the two HLE cell screenings are shown in Tables 5 and 6.

[0275] As shown in Table 5, at a dosage of 50 nM, 56 siRNAs achieved C3 mRNA inhibition rates exceeding 70%, with some sequences achieving inhibition rates exceeding 80% or even exceeding 90%. To further evaluate more promising sequences, 36 sequences with excellent inhibition rates were screened at both 10 nM and 1 nM concentrations. The results are shown in Table 6. As the dosage was further decreased, AL0161002, AL0161005, AL0161006, AL0161015, AL0161018, AL0161020, AL0161037, AL0161041, AL0161048, AL0161067, and AL0161091 showed excellent results in the dual-concentration screening, achieving inhibition rates exceeding 60% at 10 nM and approaching 60% at 1 nM.

[0276] Table 5 Knockdown levels of naked C3 siRNA sequences in HLE cells

[0277] Table 6 C3 siRNA naked sequence knockdown level in Hep3B

[0278] Example 3. In vitro activity screening of chemically modified C3-siRNA

[0279] (1) Cell culture and transfection:

[0280] (i) The culture and transfection of Hep3B cells were the same as in Example 2.

[0281] (ii) Culture and transfection of cynomolgus monkey hepatocytes

[0282] Cynomolgus macaque hepatocytes (cmTCSC, Beijing Red Biotech Co., Ltd.) were used. Preheat the culture medium. Remove the thawing medium (HEPO24, Beijing Red Biotech Co., Ltd.) from the biosafety cabinet. Add 4 mL of FBS to 36 mL of thawing medium (HEPO24) to prepare complete thawing medium. Warm the medium in a 37°C water bath for 10 minutes. Treat the cells with coating medium (HEPO44, Beijing Red Biotech Co., Ltd.) in a CO2 incubator at 37°C for 0.5 h. Remove the cells from liquid nitrogen and thaw them in a 37°C water bath. After approximately 2 minutes, remove the cells and transfer the cell suspension to 40 mL of preheated thawing medium. Rinse the cryovial with 2 mL of complete thawing medium. Centrifuge the cell suspension at 180 × g for 1 minute, discard the supernatant, and add 2 mL of preheated CM seeding medium (CMHEP054, Beijing Red Biotech Co., Ltd.). Gently pipette the cell suspension to mix thoroughly. Count 20 μl of the cell suspension. According to the counting results, 12-well plates were inoculated with 3*10 5 / well and cultured in a 37°C, 5% CO2 incubator. After 4-5 hours of attachment, the CM seeding medium was aspirated and replaced with pre-warmed culture medium (Beijing Red Biotech Co., Ltd., CMHEP064). Transfection was performed 6 hours after attachment.

[0283] Using Lipofectamine TM Transfection was performed using 3000 Transfection Reagent (thermofisher, L3000150). 50 nM modified siRNA (Suzhou Beixin Biotechnology Co., Ltd.) was diluted with 50 μl Opti-MEM (thermofisher, 1105821) for system ①, and 3 μl Lipo3000 was diluted with 50 μl Opti-MEM for system ②. After standing for 5 min, systems ① and ② were mixed and allowed to stand for another 15 min. The cells were then added dropwise to a 12-well plate. DMEM / F12 complete medium was replaced 4 h after transfection, and the 12-well plate was placed in an incubator and incubated for 48 h.

[0284] (2) RNA extraction and detection

[0285] Same as Example 2.

[0286] (3) Data statistical analysis:

[0287] Same as Example 2.

[0288] As can be seen from Table 7, in primary cynomolgus monkey hepatocytes, when the dosage was 1 nM, the inhibition rate of C3 mRNA by all sequences except AL0165002 and AL0165008 could reach 60%. In particular, the inhibition rate of C3 mRNA by sequences AL0165005, AL0165006, AL0165009, AL0165010 and AL0165011 could reach 70%, or even close to 80%.

[0289] As shown in Table 8, in Hep3B cells, when the dosage was 10 nM, AL0165017, AL0165023, AL0165031, AL0165048, AL0165062, AL0165074, AL0165082, AL0165088, AL0165107, AL0165112, AL0165114, AL0165115, AL0165116, AL0165123, AL0165124, AL0165126, AL0165128, AL0165129, AL0165134, AL0165136, AL0165143, AL0165146, AL0165149, AL0165150, AL0165151, AL0165152, AL0165154, AL0165155, AL0165156, AL0165161, and AL0165164 showed inhibition rates of greater than 80% and even approaching 90% against C3 mRNA. Furthermore, at a 1 nM dose, inhibition of C3 mRNA reached at least 60%. Modification of sequences with superior efficacy by partial base mismatching or by the addition of VPUm further enhanced their efficacy, as shown in Tables 9 and 10. The sequences with better efficacy were further tested in primary cynomolgus monkey hepatocytes. Each batch of experiments was compared with AL0165001 to screen out the sequences with better efficacy, as shown in Tables 11-13: AL0165048, AL0165088, AL0165112, AL0165116, AL0165124, AL0165129, AL0165136, AL0165146, AL0165167, AL0165171, AL0165177, AL0165178, AL0165188, and AL0165190. 65189, AL0165191, AL0165192, AL0165193, AL0165195, AL0165205, AL0165207, AL0165210, AL0165221, AL0165223, AL0165225, AL0165227, AL0165229, AL0165231, AL0165232, AL0165233, AL0165234, AL0165235, AL0165236, AL0165237 and AL0165238 performed better.

[0290] In another set of in vitro activity screening experiments on Hep3B, the results, as shown in Table 14, indicate that when the dosage is 1 nM and 0.1 nM, the efficacy of the differently modified sequences can be further enhanced. For example, the inhibition rates of AL0165242, AL0165217, AL0165246, AL0165218, AL0165247, and AL0165248 against C3 mRNA can exceed 85% or even exceed 90%. In particular, when the antisense strand contains a 2'-5'-phosphodiester bond or a substitution modification at position Im, the efficacy can be improved.

[0291] In another set of in vitro activity screening experiments in primary monkey hepatocytes, the results are shown in Table 15. When the dosage is 1 nM and 0.1 nM, the efficacy of the differently modified sequences can be further enhanced. For example, the inhibition rate of AL0165242, AL0165217, AL0165246, AL0165218, AL0165247, and AL0165248 against C3 mRNA can exceed 85% or even exceed 90%. In particular, when the antisense chain contains a 2'-5'-phosphodiester bond modification or an Im substitution modification at some positions, the efficacy can be improved.

[0292] Table 7 Knockdown levels of chemically modified C3 siRNA in primary cynomolgus monkey hepatocytes

[0293] Table 8 Knockdown levels of chemically modified C3-siRNA in Hep3B

[0294] Table 9 Knockdown levels of chemically modified C3-siRNA in Hep3B

[0295] Table 10 Knockdown levels of chemically modified C3-siRNA in Hep3B

[0296] Table 11 Knockdown levels of chemically modified C3 siRNA in primary cynomolgus monkey hepatocytes

[0297] Table 12 Knockdown levels of chemically modified C3 siRNA in primary cynomolgus monkey hepatocytes

[0298] Table 13 Knockdown levels of chemically modified C3 siRNA in primary cynomolgus monkey hepatocytes

[0299] Table 14 Knockdown levels of chemically modified C3 siRNA in Hep3B cells

[0300] Table 15 Knockdown levels of chemically modified C3 siRNA in primary monkey hepatocytes

[0301] Example 4. In vivo testing of C3 RNAi agents in C3 AAV transgenic mice

[0302] Gluc is easily secreted and highly sensitive, and can directly measure Gluc expression activity in whole blood. Gluc is connected to exogenous genes through P2A, and the expression level of Gluc directly reflects the exogenous gene mRNA level. The mRNA sequence of human C3 gene (NM_000064.3) was obtained from the NCBI database, and recombinant plasmids were obtained by molecular biology techniques such as conventional enzyme digestion and ligation. The 93-2893 fragment (HsC3_P1) and the 2293-4531 fragment (HsC3_P2) in the C3 mRNA sequence were respectively secreted and expressed as fusion proteins with Gaussia secretory luciferase (Gluc) (the gene sequence of its fusion protein is shown in SEQ ID NO.1022 and SEQ ID NO.1023). First, the sequence containing the CAG promoter (SEQ ID NO.1024) was inserted between EcoRI and XhoI of the pFB vector (purchased from Agilent, product number 013001) to obtain pFB-AAV-CAG. Then, the gene sequences seq2 and seq3 of the fusion protein were inserted between EcoRI and BamHI of the pFB-AAV-CAG vector by conventional enzyme digestion and ligation and other molecular biological techniques to obtain RA177pFB-AAV-CAG-Gluc-2A-HsC3_P1 (Figure 3) and RA178pFB-AAV-CAG-Gluc-2A-HsC3_P2 (Figure 4) vectors. The two plasmids were transfected into Sf9 cells (ATCC CRL-1711 TM ), AAV viruses 146-177 and 146-178 containing the hC3 gene were obtained and then injected through the tail vein and administered at least 29 days before the administration of C3 RNAi agents or controls.

[0303] The experiment used SPF male C57BL / 6 mice aged 6 to 8 weeks (Beijing Biotechnology Co., Ltd.). Each mouse was injected with 1.00E+12 vg / mL 146-177 or 146-178 AAV virus particles through the tail vein. Orbital blood was collected 14 days after injection, and serum was separated for Gluc luminescence detection (Pierce TMThe cells were randomly divided into two groups according to the C3-Gluc luminescence detection value: vehicle control group (NC group) and test group (AL0167001, AL0167002, AL0167003, AL0167004, AL0167005, AL0167006, AL0167007, AL0167008, AL0167009, AL0167010, AL0167011, AL0167012, AL0167013, AL0167014 and AL0167015), for a total of 15 groups. In Figure 5A , groups AL0167001, AL0167002, AL0167003, AL0167004, AL0167005, AL0167006, AL0167007, AL0167008, AL0167009, AL0167012, and AL0167013 were injected with 146-177 AAV virus; in Figure 5B , groups AL0167001, AL0167008, AL0167009, AL0167010, AL0167011, AL0167014, and AL0167015 were injected with 146-178 AAV virus, for a total of 17 groups, each containing 5 mice. After grouping, mice were subcutaneously administered a single dose of the corresponding C3 RNAi agent or a vehicle control. Blood was collected from mice before administration and on days 8, 15, and 22 after administration. Serum was separated and analyzed for luciferase luminescence to evaluate the inhibitory effect of siRNA on exogenous gene mRNA. The results are shown in Figure 5. The groups with the most significant C3 inhibitory effects were AL0167002, AL0167006, AL0167009, AL0167013, and AL0167015.

[0304] Example 5. In vivo testing of C3 RNAi agents in transgenic mice

[0305] The experiment involved SPF male hC3, NM-HU-2000079 mice (purchased from Shanghai Model Organisms), 8-9 weeks old. Pre-dose serum samples were obtained on day 0 of treatment, and the mice were randomly divided into groups based on hC3 levels. hC3, NM-HU-2000079 mice were subcutaneously administered a single 3 mg / kg dose of the C3 RNAi agents AL0167001, AL0167006, AL0167009, AL0167013, and AL0167015. Blood samples were collected from the eyeballs and analyzed within 1 hour of treatment at 1, 2, 3, 4, and 5 weeks after treatment to assess hC3 expression levels, with pre-dose control used as the control. During the experiment, no animals showed signs of mortality or moribundity. Clinical observation revealed no significant abnormalities in any of the animals. Changes in hC3 levels are shown in Figure 6A.

[0306] In vivo activity was tested in two additional groups of transgenic mice. hC3, NM-HU-2000079 mice were subcutaneously administered a single 3 mg / kg dose of a C3 RNAi agent. One group received AL0167016, AL0167017, AL0167018, AL0167019, AL0167020, AL0167021, and AL0167022, while the other group received AL0167023, AL0167024, AL0167025, and AL0167026. Blood samples were collected from the eyeballs of the mice and analyzed within 1 hour after administration. hC3 expression levels were measured, with the pre-dose control used as the control. During the experimental period, no animals showed signs of mortality or dying. Clinical observations revealed no significant abnormalities in any of the animals. The changes in hC3 levels are shown in Figures 6B and 6C.

[0307] Figure 6 shows that, compared to pre-dose levels, the knockdown effect of all C3 siRNAs reached its lowest on day 7 after drug intervention, then gradually recovered on day 14, with blood hC3 levels remaining significantly reduced through day 35. No drug-related deaths occurred in any of the test groups, and drug intervention significantly reduced hC3 levels in the mice's blood. All test groups achieved varying degrees of knockdown, ranging from 50% to 70%, compared to the vehicle control group (NC group).

Claims

1. An oligonucleotide for inhibiting the expression of complement component C3, or a pharmaceutically acceptable salt thereof, the oligonucleotide comprising a sense strand and an antisense strand, the sense strand having at least 80% sequence identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, to a sequence as shown in any one of SEQ ID NOs. 1-49 and 51-305, or a fragment thereof, or a modified sequence thereof; and the antisense strand having at least 80% sequence identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, to a sequence as shown in any one of SEQ ID NOs. 306-586, 588-586, and 1002-1009, or a fragment thereof, or a modified sequence thereof.

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein The oligonucleotide or a pharmaceutically acceptable salt thereof is selected from carboxylates, alkali metal salts, ammonium salts, alkaline earth metal salts, salts formed with organic bases and other pharmaceutically acceptable salts; Preferably, the salt is an alkali metal salt, more preferably a sodium salt or a potassium salt; Preferably, the salt is an alkaline earth metal salt, more preferably a magnesium salt or a calcium salt; Preferably, the salt is an ammonium salt, more preferably a triethylamine salt.

3. The oligonucleotide according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein The oligonucleotide comprises at least one modified nucleotide; Preferably, the oligonucleotide comprises at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotides are selected from one or more of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-fluoro modified nucleotides, and 2'-deoxy nucleotides; Preferably, the 2'-modification is a modification selected from the group consisting of: 2'-methoxy, 2'-acetylamino, 2'-aminoethyl, 2'-fluoro, 2'-O-methoxyethyl; Preferably, the oligonucleotide has a 5'-phosphate analogue modified nucleotide at the 5' end; preferably, the 5'-phosphate analogue modified nucleotide has a vinyl phosphonate modified nucleotide shown in formula (I), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl, and Base represents a nucleic acid base selected from A, G, C, T and U; preferably, the 5'-phosphate analogue modified nucleotide has a vinyl phosphate modified nucleotide shown in formula (II), wherein R is selected from H, OH, fluorine, 2'-methoxy, 2'-acetylamino, 2'-aminoethyl and 2'-O-methoxyethyl; more preferably, the 5'-phosphonate analogue modified nucleotide is APU shown in formula (III) or VPUm shown in formula (IV); Preferably, the oligonucleotide comprises a 6-(3-(2-carboxyethyl)phenyl)purine modified nucleotide; preferably, the oligonucleotide comprises formula M, which is a 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide represented by formula (V); Preferably, the oligonucleotide comprises a uridine-2'-phosphate (U-2'5') represented by formula (VI), a guanosine-2'-phosphate (G-2'5') represented by formula (VII); a cytidine-2'-phosphate (C-2'5') represented by formula (VIII); adenosine-2'-phosphate (A-2'5') represented by formula (IX) and a thymidine-2'-phosphate (T-2'5') represented by formula (X); 4. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: The oligonucleotide comprises at least one modified internucleotide linkage; Preferably, said at least one modified internucleotide linkage is a phosphorothioate linkage.

5. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: The sense strand is selected from the unmodified oligonucleotide of any one of SEQ ID NO. 2, 5, 6, 15, 18, 20, 37, 41, 48, 67, 81, 91, 115, 117, 119, 125, 133, 163, 175, 183, 189, 208, 213, 215, 216, 217, 224, 225, 227, 229, 230, 235, 237, 244, 247, 250, 251, 252, 253, 255, 256, 257, 264, 267, 271, 277, 278, 288, 289, 291, 302, 303, or the modified oligonucleotide of any one of SEQ ID NO. 588-791; the antisense strand is selected from the unmodified oligonucleotide of any one of SEQ ID NO. 307, 310, 311, 320, 323, 325, 342, 346, 353, 372, 386, 396, 420, 422, 424, 430, 438, 468, 480, 488, 494, 513, 518, 520, 521, 522, 529, 530, 532, 534, 535, 540, 542, 549, 552, 555, 556, 557, 558, 560, 561, 562, 569, 572, 573, 574, 575, 576, 578, 579, 580, 581, 582, 584, 585, 1009, or an unmodified oligonucleotide of any one of SEQ ID The modified oligonucleotide according to any one of NOs. 793-1001 and 1010-1021.

6. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: The oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand comprises the sequence shown in SEQ ID NO. 2, and the antisense strand comprises the sequence shown in SEQ ID NO. 307; (2) the sense strand comprises the sequence shown in SEQ ID NO. 5, and the antisense strand comprises the sequence shown in SEQ ID NO. 310; (3) the sense strand comprises the sequence shown in SEQ ID NO. 6, and the antisense strand comprises the sequence shown in SEQ ID NO. 311; (4) the sense strand comprises the sequence shown in SEQ ID NO. 15, and the antisense strand comprises the sequence shown in SEQ ID NO. 320; (5) the sense strand comprises the sequence shown in SEQ ID NO. 18, and the antisense strand comprises the sequence shown in SEQ ID NO. 323; (6) the sense strand comprises the sequence shown in SEQ ID NO. 37, and the antisense strand comprises the sequence shown in SEQ ID NO. 342; (7) the sense strand comprises the sequence shown in SEQ ID NO. 41, and the antisense strand comprises the sequence shown in SEQ ID NO. 346; (8) the sense strand comprises the sequence shown in SEQ ID NO. 48, and the antisense strand comprises the sequence shown in SEQ ID NO. 353; (9) the sense strand comprises the sequence shown in SEQ ID NO. 67, and the antisense strand comprises the sequence shown in SEQ ID NO. 372; (10) the sense strand comprises the sequence shown in SEQ ID NO. 91, and the antisense strand comprises the sequence shown in SEQ ID NO. 396; (11) the sense strand comprises the sequence shown in SEQ ID NO. 117, and the antisense strand comprises the sequence shown in SEQ ID NO. 422; (12) the sense strand comprises the sequence shown in SEQ ID NO. 81, and the antisense strand comprises the sequence shown in SEQ ID NO. 386; (13) the sense strand comprises the sequence shown in SEQ ID NO. 189, and the antisense strand comprises the sequence shown in SEQ ID NO. 494; (14) the sense strand comprises the sequence shown in SEQ ID NO. 213, and the antisense strand comprises the sequence shown in SEQ ID NO. 518; (15) the sense strand comprises the sequence shown in SEQ ID NO. 217, and the antisense strand comprises the sequence shown in SEQ ID NO. 522; (16) the sense strand comprises the sequence shown in SEQ ID NO. 225, and the antisense strand comprises the sequence shown in SEQ ID NO. 530; (17) the sense strand comprises the sequence shown in SEQ ID NO. 230, and the antisense strand comprises the sequence shown in SEQ ID NO. 535; (18) the sense strand comprises the sequence shown in SEQ ID NO. 237, and the antisense strand comprises the sequence shown in SEQ ID NO. 542; (19) the sense strand comprises the sequence shown in SEQ ID NO. 247, and the antisense strand comprises the sequence shown in SEQ ID NO. 552; (20) the sense strand comprises the sequence shown in SEQ ID NO. 267, and the antisense strand comprises the sequence shown in SEQ ID NO. 572; (21) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 572; (22) the sense strand comprises the sequence shown in SEQ ID NO. 277, and the antisense strand comprises the sequence shown in SEQ ID NO. 573; (23) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 574; (24) the sense strand comprises the sequence shown in SEQ ID NO. 288, and the antisense strand comprises the sequence shown in SEQ ID NO. 575; (25) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 576; (26) the sense strand comprises the sequence shown in SEQ ID NO. 115, and the antisense strand comprises the sequence shown in SEQ ID NO. 420; (27) the sense strand comprises the sequence shown in SEQ ID NO. 291, and the antisense strand comprises the sequence shown in SEQ ID NO. 578; (28) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 579; (29) the sense strand comprises the sequence shown in SEQ ID NO. 302, and the antisense strand comprises the sequence shown in SEQ ID NO. 584; (30) the sense strand comprises the sequence shown in SEQ ID NO. 303, and the antisense strand comprises the sequence shown in SEQ ID NO. 585; (31) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 582; (32) the sense strand comprises the sequence shown in SEQ ID NO. 277, and the antisense strand comprises the sequence shown in SEQ ID NO. 580; (33) the sense strand comprises the sequence shown in SEQ ID NO. 288, and the antisense strand comprises the sequence shown in SEQ ID NO. 581; (34) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 1005; (35) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 1009; Preferably, the oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand comprises the sequence shown in SEQ ID NO. 2, and the antisense strand comprises the sequence shown in SEQ ID NO. 307; (2) the sense strand comprises the sequence shown in SEQ ID NO. 18, and the antisense strand comprises the sequence shown in SEQ ID NO. 323; (3) the sense strand comprises the sequence shown in SEQ ID NO. 48, and the antisense strand comprises the sequence shown in SEQ ID NO. 353; (4) the sense strand comprises the sequence shown in SEQ ID NO. 115, and the antisense strand comprises the sequence shown in SEQ ID NO. 420; (5) the sense strand comprises the sequence shown in SEQ ID NO. 117, and the antisense strand comprises the sequence shown in SEQ ID NO. 422; (6) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 574; (7) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 579; (8) the sense strand comprises the sequence shown in SEQ ID NO. 302, and the antisense strand comprises the sequence shown in SEQ ID NO. 584; (9) the sense strand comprises the sequence shown in SEQ ID NO. 303, and the antisense strand comprises the sequence shown in SEQ ID NO. 585; (10) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 582; (11) the sense strand comprises the sequence shown in SEQ ID NO. 271, and the antisense strand comprises the sequence shown in SEQ ID NO. 572; (12) the sense strand comprises the sequence shown in SEQ ID NO. 289, and the antisense strand comprises the sequence shown in SEQ ID NO. 576; (13) the sense strand comprises the sequence shown in SEQ ID NO. 278, and the antisense strand comprises the sequence shown in SEQ ID NO. 1009; wherein each strand is independently 19 to 25 nucleotides in length.

7. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein The oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand comprises the sequence shown in SEQ ID NO. 588, and the antisense strand comprises the sequence shown in SEQ ID NO. 793; (2) the sense strand comprises the sequence shown in SEQ ID NO. 589, and the antisense strand comprises the sequence shown in SEQ ID NO. 794; (3) the sense strand comprises the sequence shown in SEQ ID NO. 590, and the antisense strand comprises the sequence shown in SEQ ID NO. 795; (4) the sense strand comprises the sequence shown in SEQ ID NO. 591, and the antisense strand comprises the sequence shown in SEQ ID NO. 796; (5) the sense strand comprises the sequence shown in SEQ ID NO. 592, and the antisense strand comprises the sequence shown in SEQ ID NO. 797; (6) the sense strand comprises the sequence shown in SEQ ID NO. 593, and the antisense strand comprises the sequence shown in SEQ ID NO. 798; (7) the sense strand comprises the sequence shown in SEQ ID NO. 594, and the antisense strand comprises the sequence shown in SEQ ID NO. 799; (8) the sense strand comprises the sequence shown in SEQ ID NO. 595, and the antisense strand comprises the sequence shown in SEQ ID NO. 800; (9) the sense strand comprises the sequence shown in SEQ ID NO. 596, and the antisense strand comprises the sequence shown in SEQ ID NO. 801; (10) the sense strand comprises the sequence shown in SEQ ID NO. 597, and the antisense strand comprises the sequence shown in SEQ ID NO. 802; (11) the sense strand comprises the sequence shown in SEQ ID NO. 599, and the antisense strand comprises the sequence shown in SEQ ID NO. 804; (12) the sense strand comprises the sequence shown in SEQ ID NO. 601, and the antisense strand comprises the sequence shown in SEQ ID NO. 806; (13) the sense strand comprises the sequence shown in SEQ ID NO. 634, and the antisense strand comprises the sequence shown in SEQ ID NO. 839; (14) the sense strand comprises the sequence shown in SEQ ID NO. 674, and the antisense strand comprises the sequence shown in SEQ ID NO. 879; (15) the sense strand comprises the sequence shown in SEQ ID NO. 698, and the antisense strand comprises the sequence shown in SEQ ID NO. 903; (16) the sense strand comprises the sequence shown in SEQ ID NO. 702, and the antisense strand comprises the sequence shown in SEQ ID NO. 907; (17) the sense strand comprises the sequence shown in SEQ ID NO. 710, and the antisense strand comprises the sequence shown in SEQ ID NO. 915; (18) the sense strand comprises the sequence shown in SEQ ID NO. 715, and the antisense strand comprises the sequence shown in SEQ ID NO. 920; (19) the sense strand comprises the sequence shown in SEQ ID NO. 722, and the antisense strand comprises the sequence shown in SEQ ID NO. 927; (20) the sense strand comprises the sequence shown in SEQ ID NO. 732, and the antisense strand comprises the sequence shown in SEQ ID NO. 937; (21) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 957; (22) the sense strand comprises the sequence shown in SEQ ID NO. 756, and the antisense strand comprises the sequence shown in SEQ ID NO. 957; (23) the sense strand comprises the sequence shown in SEQ ID NO. 762, and the antisense strand comprises the sequence shown in SEQ ID NO. 958; (24) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 959; (25) the sense strand comprises the sequence shown in SEQ ID NO. 773, and the antisense strand comprises the sequence shown in SEQ ID NO. 969; (26) the sense strand comprises the sequence shown in SEQ ID NO. 774, and the antisense strand comprises the sequence shown in SEQ ID NO. 970; (27) the sense strand comprises the sequence shown in SEQ ID NO. 776, and the antisense strand comprises the sequence shown in SEQ ID NO. 972; (28) the sense strand comprises the sequence shown in SEQ ID NO. 776, and the antisense strand comprises the sequence shown in SEQ ID NO. 973; (29) the sense strand comprises the sequence shown in SEQ ID NO. 595, and the antisense strand comprises the sequence shown in SEQ ID NO. 974; (30) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 979; (31) the sense strand comprises the sequence shown in SEQ ID NO. 756, and the antisense strand comprises the sequence shown in SEQ ID NO. 987; (32) the sense strand comprises the sequence shown in SEQ ID NO. 762, and the antisense strand comprises the sequence shown in SEQ ID NO. 989; (33) the sense strand comprises the sequence shown in SEQ ID NO. 773, and the antisense strand comprises the sequence shown in SEQ ID NO. 991; (34) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 987; (35) the sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 993; (36) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 995; (37) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 996; (38) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 997; (39) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 998; (40) the sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 999; (41) the sense strand comprises the sequence shown in SEQ ID NO. 756, and the antisense strand comprises the sequence shown in SEQ ID NO. 996; (42) the sense strand comprises the sequence shown in SEQ ID NO. 762, and the antisense strand comprises the sequence shown in SEQ ID NO. 1000; (43) the sense strand comprises the sequence shown in SEQ ID NO. 773, and the antisense strand comprises the sequence shown in SEQ ID NO. 1001; (44) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 1013; (45) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 1017; (46) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 1018; (47) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 1021; (48) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 985; Preferably, the oligonucleotide comprises any one selected from the following sense strand and antisense strand combinations: (1) the sense strand comprises the sequence shown in SEQ ID NO. 588, and the antisense strand comprises the sequence shown in SEQ ID NO. 793; (2) the sense strand comprises the sequence shown in SEQ ID NO. 592, and the antisense strand comprises the sequence shown in SEQ ID NO. 797; (3) the sense strand comprises the sequence shown in SEQ ID NO. 595, and the antisense strand comprises the sequence shown in SEQ ID NO. 800; (4) the sense strand comprises the sequence shown in SEQ ID NO. 599, and the antisense strand comprises the sequence shown in SEQ ID NO. 804; (5) the sense strand comprises the sequence shown in SEQ ID NO. 601, and the antisense strand comprises the sequence shown in SEQ ID NO. 806; (6) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 995; (7) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 996; (8) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 997; (9) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 998; (10) the sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 999; (11) the sense strand comprises the sequence shown in SEQ ID NO. 752, and the antisense strand comprises the sequence shown in SEQ ID NO. 987; (12) the sense strand comprises the sequence shown in SEQ ID NO. 786, and the antisense strand comprises the sequence shown in SEQ ID NO. 993; (13) the sense strand comprises the sequence shown in SEQ ID NO. 788, and the antisense strand comprises the sequence shown in SEQ ID NO. 1017; (14) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 1018; (15) the sense strand comprises the sequence shown in SEQ ID NO. 763, and the antisense strand comprises the sequence shown in SEQ ID NO. 1021; (16) the sense strand comprises the sequence shown in SEQ ID NO. 789, and the antisense strand comprises the sequence shown in SEQ ID NO. 985; wherein each strand is independently 19 to 25 nucleotides in length.

8. A conjugate for inhibiting the expression of complement component C3 or a pharmaceutically acceptable salt thereof, comprising: (i) the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, and (ii) a targeting ligand conjugated to the oligonucleotide or a pharmaceutically acceptable salt thereof, wherein: At least one nucleotide of the oligonucleotide is conjugated to a targeting ligand; Preferably, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide or a lipid; Preferably, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety; Preferably, the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety or a tetravalent GalNAc moiety; Preferably, the targeting ligand is L96; 9. A composition comprising the oligonucleotide according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, or the conjugate according to claim 8 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier; Preferably, the composition is in the form of an oral dosage form, an intravenous injection, a subcutaneous injection or an intramuscular injection; Preferably, the composition further comprises other drugs for treating and / or preventing complement component C3-related disorders.

10. The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, the conjugate or pharmaceutically acceptable salt thereof according to claim 8, or the composition according to claim 9 for use in treating and / or preventing complement component C3-related disorders; The complement component C3-associated disorder is selected from cold agglutinin disease (CAD), warm autoimmune hemolytic anemia, and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), bullous pemphigoid, pemphigus, such as pemphigus vulgaris (PV) and pemphigus foliaceus (PF), or C3 glomerulopathy.

Images