Oligonucleotides targeting complement component C3 genes and uses thereof

Abstract

Provided are an oligonucleotide targeting a complement component C3 gene, which can effectively reduce the content of complement component C3 in vivo, and which is an effective inhibitor of complement component C3, and uses thereof.

Description

Technical Field

[0001] The present disclosure relates to an oligonucleotide, particularly 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 and was discovered more than a hundred years ago. It is called complement because people found that in addition to antibodies in the body, there are some proteins that have a supplementary effect on the immune system.

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

[0004] The entire activation process of the complement system is manifested as a series of cascade enzymatic hydrolysis reactions of serine proteases, and finally forms a membrane attack complex leading to target cell damage. The activation of the complement system is mainly achieved through three relatively independent and interrelated pathways: the classical pathway, the alternative pathway, and the lectin pathway ( Figure 1 ), and the C5 convertases generated by the three pathways can all cleave C5, trigger a common terminal effect, and then exert various biological effects such as opsonophagocytosis, cell lysis, inflammation mediation, immune regulation, and clearance of immune complexes. Currently, the drug development targeting the relevant targets of the three pathways has been successfully transformed. Given the clear and specific pathogenesis of some rare diseases related to complement, rare disease patients currently benefit the most significantly.

[0005] Complement C3 is the complement component with the highest content in serum. It is located upstream of C5 and is the terminal effector molecule of multiple activation pathways of complement. Under the action of C3 convertase, complement C3 can be cleaved into C3a and C3b, playing an important role in the classical activation pathway and alternative activation pathway of complement.

[0006] Complement C5 is a representative molecule of the membrane attack complex (MAC) in the complement system and is a popular target in the drug development targeting complement. C5 inhibitors are the earliest approved complement drugs. In March 2007, the FDA approved the marketing of the complement C5 monoclonal antibody Eculizumab (Soliris) of Alexion Pharmaceuticals for the treatment of paroxysmal nocturnal hemoglobinuria (PNH).

[0007] Soliris is the world's first C5 complement inhibitor, administered by injecting Eculizumab once every two weeks. Soliris is also the first specific drug for PNH patients. Since then, Soliris has also been approved for indications such as atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), and adult neuromyelitis optica.

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

[0009] The inappropriate activation of the complement system is the cause of the spread 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. The therapies available for treating complement component C3-related diseases are limited, require time-consuming and invasive administration, and are costly. Therefore, there is a need in the art for alternative and combination therapies for subjects suffering from complement component C3-related diseases. Summary of the Invention

[0010] The object of the present disclosure is to provide an inhibitor that inhibits the expression of complement component C3 with good efficacy, high safety, and long-lasting drug effect.

[0011] The present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof and a method for using the oligonucleotide or a pharmaceutically acceptable salt thereof to inhibit the expression of complement component C3 gene in cells or mammals, wherein the oligonucleotide targets the complement component C3 gene. The present disclosure also provides a composition and a method for treating pathological conditions and diseases in mammals caused by the expression of complement component C3 gene. The oligonucleotide is double-stranded RNA (dsRNA) and directs the sequence-specific degradation of mRNA through a process called RNA interference (RNAi).

[0012] In one aspect, the present disclosure provides an oligonucleotide or a pharmaceutically acceptable salt thereof for inhibiting the expression of complement component C3 gene. The oligonucleotide includes a sense strand and an antisense strand. The sense strand has a sequence with at least 80% sequence identity to the sequence shown in any one of SEQ ID NO.SEQ IDNO.1 - 49 and 51 - 305, or a fragment thereof, or a modified sequence of the foregoing sequence or its fragment, preferably a sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity; the antisense strand has a sequence with at least 80% sequence identity to the sequence shown in any one of SEQ ID NO.306 - 586, 588 - 586 and 1002 - 1009, or a fragment thereof, or a modified sequence of the foregoing sequence or its fragment, preferably a sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0013] In another aspect, the present disclosure provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting the expression of complement component C3, which includes: (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, which includes the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or the foregoing conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

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

[0016] In another aspect, the present disclosure provides a method for treating and / or preventing complement component C3 - related diseases, diseases and / or conditions in a subject by administering a therapeutic agent (such as the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, or the foregoing conjugate or a pharmaceutically acceptable salt thereof, or the foregoing composition, or a vector or transgenic encoding the oligonucleotide) to the subject.

[0017] In another aspect, the present disclosure provides a method for treating and / or preventing complement C3 - related diseases, diseases and / or conditions in a subject by combining the foregoing oligonucleotide or a pharmaceutically acceptable salt thereof, conjugate or a pharmaceutically acceptable salt thereof, or composition with other drugs and / or other treatment methods.

[0018] Experiments have shown that the oligonucleotides of the present disclosure can effectively reduce the content of complement component C3 in vivo and are effective inhibitors of complement component C3. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The complement activation pathway is shown.

[0020] Figure 2 The flowchart of solid-phase synthesis of siRNA is shown.

[0021] Figure 3 The restriction map of RA177 pFB-AAV-CAG-Gluc-2A-HsC3_P1 is shown.

[0022] Figure 4 The restriction map of RA178 pFB-AAV-CAG-Gluc-2A-HsC3_P2 is shown.

[0023] Figure 5 The in vivo efficacy of hC3 siRNA (AAV hC3 transgenic mice) is shown.

[0024] Figure 6 The in vivo efficacy of hC3 siRNA (hC3 transgenic mice) is shown. DETAILED DESCRIPTION

[0025] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory operation procedures used herein are all terms and conventional procedures widely used in the corresponding fields. At the same time, to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

[0026] As used herein, the term "about" or "approximate" as applied to one or more target values refers to a value similar to the reference value. In certain embodiments, unless otherwise specified or otherwise obvious from the context, the term "approximate" or "about" refers to a range of values that fall 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) (unless such a number would exceed 100% of the possible value).

[0027] The present text describes RNAi agents of complement component C3 for selectively and effectively inhibiting the expression of the complement component C3 gene. The RNAi agents of complement component C3 described herein can be used for preventing or treating the following diseases or for preparing a medicament 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, a "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the complement component C3 gene, including the mRNA that is a processed product of the RNA as a primary transcript.

[0029] As used herein, the term "complementary" refers to the structural relationship between nucleotides (e.g., between two nucleotides on opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the nucleotides to form base pairs with each other. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of the opposing nucleic acid can base pair together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides can base pair in a Watson-Crick manner or in any other manner that allows for the formation of a stable duplex. In some embodiments, two nucleic acids can have nucleotide sequences that are complementary to each other so as to form a 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, a strand has two free ends, such as 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 as compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide that has one or more atoms modified or substituted (including modifications or substitutions in the sugar, phosphate group, or base or modifications or substitutions in the sugar, phosphate group, or base) other than at the 2'-position.

[0032] As used herein, the term "oligonucleotide" refers to short nucleic acids, such as short nucleic acids having a length of less than 100 nucleotides. Oligonucleotides can comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides can be single-stranded or double-stranded. Oligonucleotides can have or can lack duplex regions. As a non-limiting set of examples, oligonucleotides can be, but are not limited to, small interfering RNAs (siRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), Dicer-substrate interfering RNAs (dsiRNAs), antisense oligonucleotides, short siRNAs, or single-stranded siRNAs. 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, complementary base pairing forms one or more duplex regions of the double-stranded oligonucleotide between the antiparallel sequences of nucleotides of covalently separated nucleic acid strands. In some embodiments, complementary base pairing forms one or more duplex regions of the double-stranded oligonucleotide between the antiparallel sequences of nucleotides of covalently linked nucleic acid strands. In some embodiments, complementary base pairing forms one or more duplex regions of the double-stranded oligonucleotide from a single nucleic acid strand that is folded (e.g., via a hairpin) to provide antiparallel sequences of nucleotides that base pair together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are fully duplexed with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid strands that are partially duplexed, e.g., having overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary and, thus, can have one or more mismatches, which can include internal mismatches or terminal mismatches.

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

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

[0036] As used herein, "conjugation" refers to the joining of two or more chemical moieties, each having a specific function, to one another in a covalent linkage; accordingly, a "conjugate" refers to a compound formed by the covalent linkage between the respective chemical moieties. Further, a "siRNA conjugate" refers to a compound formed by covalently linking one or more chemical moieties having specific functions to an siRNA. Hereinafter, the siRNA conjugates of the present disclosure are sometimes also simply referred to as "conjugates". The siRNA conjugate should be understood, depending on the context, as a general term for siRNA conjugates, a first siRNA conjugate or a second siRNA conjugate, or an siRNA sense strand conjugate or an siRNA antisense strand conjugate.

[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 to the internucleotide linkage, sugar moiety, or nucleobase, such as functional groups or atoms. Modifications applicable to 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 double-stranded RNAi. For example, a nucleotide overhang exists when the 3' end of one strand of dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang can comprise or consist of the following: nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang can be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang can be present at the 5' end, 3' end, or both ends of the antisense or 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" can be used interchangeably with "knock down", "reduce", "silence", "downregulate", "suppress", and other similar terms, and includes any degree of inhibitory effect.

[0041] The phrase "inhibit the expression of complement component C3" is intended to refer to inhibiting the expression of any complement component C3 gene (such as, for example, the mouse complement component C3 gene, the rat complement component C3 gene, the monkey complement component C3 gene, or the human complement component C3 gene), as well as variants or mutants of the complement component C3 gene. Thus, in the context of genetically manipulating 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] "Inhibit the expression of the complement component C3 gene" includes inhibition of the complement component C3 gene at any level, for example, at least partial inhibition of the expression of the complement component C3 gene. The expression of the complement component C3 gene can be evaluated based on the level or change in level of any variable associated with the expression of the complement component C3 gene, such as, for example, the complement component mRNA level or the complement component C3 protein level, or by inhibiting the mRNA level of the Gluc and C3 fusion protein gene, and thereby indirectly reflecting the inhibition of the C3 protein level by inhibiting the Gluc protein level.

[0043] This level can be evaluated in individual cells or cell populations, including, for example, samples from a subject. It is understood that complement component C3 is mainly expressed in the liver, but is also expressed in the brain, gallbladder, heart, and kidneys, and is present in the circulation.

[0044] Inhibition can be evaluated 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, e.g., a pre-dose baseline level, or a level determined in similar subjects that have not been treated or have been treated with a control (such as, for example, a buffer only control or a non-active agent control).

[0045] The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the free base or free acid and 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 (especially 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, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine. In addition, these salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, alkali metal salts (such as sodium, potassium and lithium salts), ammonium salts, alkaline earth metal salts (such as calcium 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, polyamine resins. The oligonucleotides of the present disclosure can also exist in zwitterionic form. 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" is an animal that endogenously or heterologously expresses a target gene, such as a mammal, including primates (such as humans, non-human primates, e.g., 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 a reduction in at least one sign or symptom of a complement component C3-related disorder in a subject. Treatment also includes reducing one or more signs or symptoms associated with undesired complement component C3 expression; reducing the degree of undesired complement component C3 activation or stabilization; improving or alleviating undesired complement component C3 activation or stabilization. Treatment also includes reducing one or more signs or symptoms associated with undesired complement component C3 expression. "Treatment" can also mean prolonging survival compared to the expected survival without treatment.

[0048] As used herein, the terms "prevention" or "preventing", when used in reference to a disease or disorder, will 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 the amount of an RNAi agent that, when administered to a subject having a complement component C3-related disorder, is sufficient to affect the treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the subject to be treated.

[0050] As used herein, the term "preventively effective amount" is intended to encompass the amount of an RNAi agent that, when administered to a subject having a complement component C3-related disorder, is sufficient to prevent or ameliorate the disorder or one or more symptoms of the disorder. Ameliorating a disease includes slowing the progression of the disease or reducing the severity of a disease that develops later. The "preventively effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of disease risk, and the medical history, age, weight, family history, genetic makeup, type of prior treatment or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

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

[0052] In some embodiments of the present disclosure, 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 5' and / or 3'-overhang sequences that are one or more nucleotides in length, wherein the 5' and / or 3'-overhang sequences are present on the antisense strand and / or the sense strand. In one embodiment, the overhang at the 3'-end or 5'-end of the antisense strand of the oligonucleotide has 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the overhang at the 3'-end or 5'-end of the sense strand of the dsRNA has 1 to 10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside phosphorothioates.

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

[0057] In some embodiments of the present disclosure, the sense strand bears 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: 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 having a length in the range of 19 to 23 nucleotides.

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

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

[0064] In some embodiments of the present disclosure, the oligonucleotide comprises a 5' overhang of 1 nucleotide in length and a 3'-overhang sequence of 1 nucleotide in length, wherein the 5' overhang and the 3'-overhang sequence are present on the antisense strand, and wherein the sense strand has a length of 19 nucleotides and the antisense strand has a length of 21 nucleotides, 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 of 2 nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand has a length of 19 nucleotides and the antisense strand has a length of 21 nucleotides, such that the sense strand and the antisense strand form a duplex of 19 nucleotides in length.

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

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

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

[0069] In some embodiments of the present disclosure, a 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 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, 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), and metal salts such as sodium salts, potassium salts, and magnesium salts, etc.

[0071] In some embodiments of the present disclosure, the oligonucleotide or its salt 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 nucleotide is 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'-deoxynucleotides.

[0075] In some embodiments of the present disclosure, the 2'-modification is a modification selected from the following: 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 vinylphosphonate-modified nucleotide represented by formula (I), wherein R is selected from H, OH, fluoro, 2'-methoxy, 2'-acetamido, 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 represented by formula (II), wherein R is selected from H, OH, fluoro, 2'-methoxy, 2'-acetamido, 2'-aminoethyl, and 2'-O-methoxyethyl; more preferably, the 5'-phosphonate analog-modified nucleotide is APU represented by formula (III) or VPUm represented by formula (IV);

[0077]

[0078] 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 represented by formula (V);

[0079] M

[0080]

[0081] In some embodiments of the present disclosure, the oligonucleotide comprises a uridine-2'-phosphate (U-2'5') selected from formula (VI), a guanosine-2'-phosphate (G-2'5') selected from formula (VII); a cytidine-2'-phosphate (C-2'5') selected from formula (VIII); an adenosine-2'-phosphate (A-2'5') selected from formula (IX), and a thymidine-2'-phosphate (T-2'5') selected from formula (X));

[0082]

[0083] In some embodiments of the present disclosure, the oligonucleotide comprises at least one modified internucleotide bond.

[0084] In some embodiments of the present disclosure, at least one modified internucleotide bond is a phosphorothioate bond. The phosphorothioate internucleotide bond modification can occur at any position in the strand, on the sense strand, the antisense strand, or any nucleotide of both strands. For example, the internucleotide bond modification can occur on each nucleotide of the sense strand or the antisense strand; each internucleotide bond modification can occur in an alternating pattern on the sense strand or the antisense strand; or the sense strand or the antisense strand can contain two internucleotide bond modifications in an alternating pattern. The alternating pattern of internucleotide bond modification on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of internucleotide bond modification on the sense strand can be offset relative to the alternating pattern of internucleotide bonds on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 4 to 8 phosphorothioate internucleotide bonds. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide bonds at the 5' end and two phosphorothioate internucleotide bonds at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide bonds at the 5' end or the 3' end.

[0085] In some embodiments of the present disclosure, the sense strand is selected from any of the unmodified oligonucleotides set forth in 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 any of the modified oligonucleotides set forth in SEQ ID NO.588 - 791; the antisense strand is selected from any of the unmodified oligonucleotides set forth in 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 any of the modified oligonucleotides set forth in SEQ ID NO.793 - 1001, 1010 - 1021.

[0086] In some embodiments of the present disclosure, the oligonucleotide comprises any one of the following combinations of sense and antisense strands:

[0087] (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;

[0088] (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;

[0089] (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;

[0090] (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;

[0091] (5) The sense strand contains the sequence shown in SEQ ID NO. 18, and the antisense strand contains the sequence shown in SEQ ID NO. 323;

[0092] (6) The sense strand contains the sequence shown in SEQ ID NO. 37, and the antisense strand contains the sequence shown in SEQ ID NO. 342;

[0093] (7) The sense strand contains the sequence shown in SEQ ID NO. 41, and the antisense strand contains the sequence shown in SEQ ID NO. 346;

[0094] (8) The sense strand contains the sequence shown in SEQ ID NO. 48, and the antisense strand contains the sequence shown in SEQ ID NO. 353;

[0095] (9) The sense strand contains the sequence shown in SEQ ID NO. 67, and the antisense strand contains the sequence shown in SEQ ID NO. 372;

[0096] (10) The sense strand contains the sequence shown in SEQ ID NO. 91, and the antisense strand contains the sequence shown in SEQ ID NO. 396;

[0097] (11) The sense strand contains the sequence shown in SEQ ID NO. 117, and the antisense strand contains the sequence shown in SEQ ID NO. 422;

[0098] (12) The sense strand contains the sequence shown in SEQ ID NO. 81, and the antisense strand contains the sequence shown in SEQ ID NO. 386;

[0099] (13) The sense strand contains the sequence shown in SEQ ID NO. 189, and the antisense strand contains the sequence shown in SEQ ID NO. 494;

[0100] (14) The sense strand contains the sequence shown in SEQ ID NO. 213, and the antisense strand contains the sequence shown in SEQ ID NO. 518;

[0101] (15) The sense strand contains the sequence shown in SEQ ID NO. 217, and the antisense strand contains the sequence shown in SEQ ID NO. 522;

[0102] (16) The sense strand contains the sequence shown in SEQ ID NO. 225, and the antisense strand contains the sequence shown in SEQ ID NO. 530;

[0103] (17) The sense strand contains the sequence shown in SEQ ID NO. 230, and the antisense strand contains the sequence shown in SEQ ID NO. 535;

[0104] (18) The sense strand contains the sequence shown in SEQ ID NO. 237, and the antisense strand contains the sequence shown in SEQ ID NO. 542;

[0105] (19) The sense strand contains the sequence shown in SEQ ID NO. 247, and the antisense strand contains the sequence shown in SEQ ID NO. 552;

[0106] (20) The sense strand contains the sequence shown in SEQ ID NO. 267, and the antisense strand contains the sequence shown in SEQ ID NO. 572;

[0107] (21) The sense strand contains the sequence shown in SEQ ID NO. 271, and the antisense strand contains the sequence shown in SEQ ID NO. 572;

[0108] (22) The sense strand contains the sequence shown in SEQ ID NO. 277, and the antisense strand contains the sequence shown in SEQ ID NO. 573;

[0109] (23) The sense strand contains the sequence shown in SEQ ID NO. 278, and the antisense strand contains the sequence shown in SEQ ID NO. 574;

[0110] (24) The sense strand contains the sequence shown in SEQ ID NO. 288, and the antisense strand contains the sequence shown in SEQ ID NO. 575;

[0111] (25) The sense strand contains the sequence shown in SEQ ID NO. 289, and the antisense strand contains the sequence shown in SEQ ID NO. 576;

[0112] (26) The sense strand contains the sequence shown in SEQ ID NO. 115, and the antisense strand contains the sequence shown in SEQ ID NO. 420;

[0113] (27) The sense strand contains the sequence shown in SEQ ID NO. 291, and the antisense strand contains the sequence shown in SEQ ID NO. 578;

[0114] (28) The sense strand contains the sequence shown in SEQ ID NO. 271, and the antisense strand contains the sequence shown in SEQ ID NO. 579;

[0115] (29) The sense strand contains the sequence shown in SEQ ID NO. 302, and the antisense strand contains the sequence shown in SEQ ID NO. 584;

[0116] (30) The sense strand contains the sequence shown in SEQ ID NO. 303, and the antisense strand contains the sequence shown in SEQ ID NO. 585;

[0117] (31) The sense strand contains the sequence shown in SEQ ID NO. 289, and the antisense strand contains the sequence shown in SEQ ID NO. 582;

[0118] (32) The sense strand contains the sequence shown in SEQ ID NO. 277, and the antisense strand contains the sequence shown in SEQ ID NO. 580;

[0119] (33) The sense strand contains the sequence shown in SEQ ID NO. 288, and the antisense strand contains the sequence shown in SEQ ID NO. 581;

[0120] (34) The sense strand contains the sequence shown in SEQ ID NO. 278, and the antisense strand contains the sequence shown in SEQ ID NO. 1005;

[0121] (35) The sense strand contains the sequence shown in SEQ ID NO. 278, and the antisense strand contains the sequence shown in SEQ ID NO. 1009;

[0122] Wherein each strand is independently 19 to 25 nucleotides in length.

[0123] In some embodiments of the present disclosure, the oligonucleotide comprises any one of the following combinations of sense and antisense strands:

[0124] (1) The sense strand contains the sequence shown in SEQ ID NO. 2, and the antisense strand contains the sequence shown in SEQ ID NO. 307;

[0125] (2) The sense strand contains the sequence shown in SEQ ID NO. 18, and the antisense strand contains the sequence shown in SEQ ID NO. 323;

[0126] (3) The sense strand contains the sequence shown in SEQ ID NO. 48, and the antisense strand contains the sequence shown in SEQ ID NO. 353;

[0127] (4) The sense strand contains the sequence shown in SEQ ID NO. 115, and the antisense strand contains the sequence shown in SEQ ID NO. 420;

[0128] (5) The sense strand contains the sequence shown in SEQ ID NO. 117, and the antisense strand contains the sequence shown in SEQ ID NO. 422;

[0129] (6) The sense strand contains the sequence shown in SEQ ID NO. 278, and the antisense strand contains the sequence shown in SEQ ID NO. 574;

[0130] (7) The sense strand contains the sequence shown in SEQ ID NO. 271, and the antisense strand contains the sequence shown in SEQ ID NO. 579;

[0131] (8) The sense strand contains the sequence shown in SEQ ID NO. 302, and the antisense strand contains the sequence shown in SEQ ID NO. 584;

[0132] (9) The sense strand contains the sequence shown in SEQ ID NO. 303, and the antisense strand contains the sequence shown in SEQ ID NO. 585;

[0133] (10) The sense strand contains the sequence shown in SEQ ID NO. 289, and the antisense strand contains the sequence shown in SEQ ID NO. 582;

[0134] (11) The sense strand contains the sequence shown in SEQ ID NO. 271, and the antisense strand contains the sequence shown in SEQ ID NO. 572;

[0135] (12) The sense strand contains the sequence shown in SEQ ID NO. 289, and the antisense strand contains the sequence shown in SEQ ID NO. 576;

[0136] (13) The sense strand contains the sequence shown in SEQ ID NO. 278, and the antisense strand contains the sequence shown in SEQ ID NO. 1009.

[0137] In some embodiments of the present disclosure, the oligonucleotide comprises any one of the following combinations of sense and antisense strands:

[0138] (1) The sense strand contains the sequence shown in SEQ ID NO. 588, and the antisense strand contains the sequence shown in SEQ ID NO. 793;

[0139] (2) The sense strand contains the sequence shown in SEQ ID NO. 589, and the antisense strand contains the sequence shown in SEQ ID NO. 794;

[0140] (3) The sense strand contains the sequence shown in SEQ ID NO. 590, and the antisense strand contains the sequence shown in SEQ ID NO. 795;

[0141] (4) The sense strand contains the sequence shown in SEQ ID NO. 591, and the antisense strand contains the sequence shown in SEQ ID NO. 796;

[0142] (5) The sense strand contains the sequence shown in SEQ ID NO. 592, and the antisense strand contains the sequence shown in SEQ ID NO. 797;

[0143] (6) The sense strand contains the sequence shown in SEQ ID NO. 593, and the antisense strand contains the sequence shown in SEQ ID NO. 798;

[0144] (7) The sense strand contains the sequence shown in SEQ ID NO. 594, and the antisense strand contains the sequence shown in SEQ ID NO. 799;

[0145] (8) The sense strand contains the sequence shown in SEQ ID NO. 595, and the antisense strand contains the sequence shown in SEQ ID NO. 800;

[0146] (9) The sense strand contains the sequence shown in SEQ ID NO. 596, and the antisense strand contains the sequence shown in SEQ ID NO. 801;

[0147] (10) The sense strand contains the sequence shown in SEQ ID NO. 597, and the antisense strand contains the sequence shown in SEQ ID NO. 802;

[0148] (11) The sense strand contains the sequence shown in SEQ ID NO. 599, and the antisense strand contains the sequence shown in SEQ ID NO. 804;

[0149] (12) The sense strand contains the sequence shown in SEQ ID NO. 601, and the antisense strand contains the sequence shown in SEQ ID NO. 806;

[0150] (13) The sense strand contains the sequence shown in SEQ ID NO. 634, and the antisense strand contains the sequence shown in SEQ ID NO. 839;

[0151] (14) The sense strand contains the sequence shown in SEQ ID NO. 674, and the antisense strand contains the sequence shown in SEQ ID NO. 879;

[0152] (15) The sense strand contains the sequence shown in SEQ ID NO. 698, and the antisense strand contains the sequence shown in SEQ ID NO. 903;

[0153] (16) The sense strand contains the sequence shown in SEQ ID NO. 702, and the antisense strand contains the sequence shown in SEQ ID NO. 907;

[0154] (17) The sense strand contains the sequence shown in SEQ ID NO. 710, and the antisense strand contains the sequence shown in SEQ ID NO. 915;

[0155] (18) The sense strand contains the sequence shown in SEQ ID NO. 715, and the antisense strand contains the sequence shown in SEQ ID NO. 920;

[0156] (19) The sense strand contains the sequence shown in SEQ ID NO. 722, and the antisense strand contains the sequence shown in SEQ ID NO. 927;

[0157] (20) The sense strand contains the sequence shown in SEQ ID NO. 732, and the antisense strand contains the sequence shown in SEQ ID NO. 937;

[0158] (21) The sense strand contains the sequence shown in SEQ ID NO. 752, and the antisense strand contains the sequence shown in SEQ ID NO. 957;

[0159] (22) The sense strand contains the sequence shown in SEQ ID NO. 756, and the antisense strand contains the sequence shown in SEQ ID NO. 957;

[0160] (23) The sense strand contains the sequence shown in SEQ ID NO. 762, and the antisense strand contains the sequence shown in SEQ ID NO. 958;

[0161] (24) The sense strand contains the sequence shown in SEQ ID NO. 763, and the antisense strand contains the sequence shown in SEQ ID NO. 959;

[0162] (25) The sense strand contains the sequence shown in SEQ ID NO. 773, and the antisense strand contains the sequence shown in SEQ ID NO. 969;

[0163] (26) The sense strand contains the sequence shown in SEQ ID NO. 774, and the antisense strand contains the sequence shown in SEQ ID NO. 970;

[0164] (27) The sense strand contains the sequence shown in SEQ ID NO. 776, and the antisense strand contains the sequence shown in SEQ ID NO. 972;

[0165] (28) The sense strand contains the sequence shown in SEQ ID NO. 776, and the antisense strand contains the sequence shown in SEQ ID NO. 973;

[0166] (29) The sense strand contains the sequence shown in SEQ ID NO. 595, and the antisense strand contains the sequence shown in SEQ ID NO. 974;

[0167] (30) The sense strand contains the sequence shown in SEQ ID NO.763, and the antisense strand contains the sequence shown in SEQ ID NO.979;

[0168] (31) The sense strand contains the sequence shown in SEQ ID NO.756, and the antisense strand contains the sequence shown in SEQ ID NO.987;

[0169] (32) The sense strand contains the sequence shown in SEQ ID NO.762, and the antisense strand contains the sequence shown in SEQ ID NO.989;

[0170] (33) The sense strand contains the sequence shown in SEQ ID NO.773, and the antisense strand contains the sequence shown in SEQ ID NO.991;

[0171] (34) The sense strand contains the sequence shown in SEQ ID NO.752, and the antisense strand contains the sequence shown in SEQ ID NO.987;

[0172] (35) The sense strand contains the sequence shown in SEQ ID NO.786, and the antisense strand contains the sequence shown in SEQ ID NO.993;

[0173] (36) The sense strand contains the sequence shown in SEQ ID NO.763, and the antisense strand contains the sequence shown in SEQ ID NO.995;

[0174] (37) The sense strand contains the sequence shown in SEQ ID NO.752, and the antisense strand contains the sequence shown in SEQ ID NO.996;

[0175] (38) The sense strand contains the sequence shown in SEQ ID NO.788, and the antisense strand contains the sequence shown in SEQ ID NO.997;

[0176] (39) The sense strand contains the sequence shown in SEQ ID NO.789, and the antisense strand contains the sequence shown in SEQ ID NO.998;

[0177] (40) The sense strand contains the sequence shown in SEQ ID NO.786, and the antisense strand contains the sequence shown in SEQ ID NO.999;

[0178] (41) The sense strand contains the sequence shown in SEQ ID NO.756, and the antisense strand contains the sequence shown in SEQ ID NO.996;

[0179] (42) The sense strand contains the sequence shown in SEQ ID NO.762, and the antisense strand contains the sequence shown in SEQ ID NO.1000;

[0180] (43) The sense strand contains the sequence shown in SEQ ID NO.773, and the antisense strand contains the sequence shown in SEQ ID NO.1001;

[0181] (44) The sense strand contains the sequence shown in SEQ ID NO.763, and the antisense strand contains the sequence shown in SEQ ID NO.1013;

[0182] (45) The sense strand contains the sequence shown in SEQ ID NO.788, and the antisense strand contains the sequence shown in SEQ ID NO.1017;

[0183] (46) The sense strand contains the sequence shown in SEQ ID NO.789, and the antisense strand contains the sequence shown in SEQ ID NO.1018;

[0184] (47) The sense strand contains the sequence shown in SEQ ID NO.763, and the antisense strand contains the sequence shown in SEQ ID NO.1021;

[0185] (48) The sense strand contains the sequence shown in SEQ ID NO.789, and the antisense strand contains the sequence shown in SEQ ID NO.985;

[0186] Wherein each strand is independently 19 to 25 nucleotides in length.

[0187] In some embodiments of the present disclosure, the oligonucleotide comprises any one of the following combinations of sense and antisense strands:

[0188] (1) The sense strand contains the sequence shown in SEQ ID NO.588, and the antisense strand contains the sequence shown in SEQ ID NO.793;

[0189] (2) The sense strand contains the sequence shown in SEQ ID NO.592, and the antisense strand contains the sequence shown in SEQ ID NO.797;

[0190] (3) The sense strand contains the sequence shown in SEQ ID NO.595, and the antisense strand contains the sequence shown in SEQ ID NO.800;

[0191] (4) The sense strand contains the sequence shown in SEQ ID NO.599, and the antisense strand contains the sequence shown in SEQ ID NO.804;

[0192] (5) The sense strand contains the sequence shown in SEQ ID NO. 601, and the antisense strand contains the sequence shown in SEQ ID NO. 806;

[0193] (6) The sense strand contains the sequence shown in SEQ ID NO. 763, and the antisense strand contains the sequence shown in SEQ ID NO. 995;

[0194] (7) The sense strand contains the sequence shown in SEQ ID NO. 752, and the antisense strand contains the sequence shown in SEQ ID NO. 996;

[0195] (8) The sense strand contains the sequence shown in SEQ ID NO. 788, and the antisense strand contains the sequence shown in SEQ ID NO. 997;

[0196] (9) The sense strand contains the sequence shown in SEQ ID NO. 789, and the antisense strand contains the sequence shown in SEQ ID NO. 998;

[0197] (10) The sense strand contains the sequence shown in SEQ ID NO. 786, and the antisense strand contains the sequence shown in SEQ ID NO. 999;

[0198] (11) The sense strand contains the sequence shown in SEQ ID NO. 752, and the antisense strand contains the sequence shown in SEQ ID NO. 987;

[0199] (12) The sense strand contains the sequence shown in SEQ ID NO. 786, and the antisense strand contains the sequence shown in SEQ ID NO. 993.

[0200] (13) The sense strand contains the sequence shown in SEQ ID NO. 788, and the antisense strand contains the sequence shown in SEQ ID NO. 1017;

[0201] (14) The sense strand contains the sequence shown in SEQ ID NO. 789, and the antisense strand contains the sequence shown in SEQ ID NO. 1018;

[0202] (15) The sense strand contains the sequence shown in SEQ ID NO. 763, and the antisense strand contains the sequence shown in SEQ ID NO. 1021;

[0203] (16) The sense strand contains the sequence shown in SEQ ID NO. 789, and the antisense strand contains the sequence shown in SEQ ID NO. 985.

[0204] The present disclosure also provides a conjugate or a pharmaceutically acceptable salt thereof for inhibiting the expression of complement component C3, 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 its salt is conjugated to a targeting ligand to form an siRNA conjugate. The aforementioned siRNA conjugate contains the above siRNA and a conjugation group conjugated to the siRNA. The term "oligonucleotidate" refers to an oligonucleotide compound in salt form. Oligonucleotidates include salts of oligonucleotide-conjugated compounds and salts of unconjugated oligonucleotide compounds. Oligonucleotidates advantageously exist in the form of solid powders.

[0205] Generally speaking, the conjugation group comprises at least one pharmaceutically acceptable targeting ligand and an optional linker, and the siRNA, the linker, and the targeting ligand are connected in sequence. The targeting group can be a ligand conventionally used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, there are 2-4 targeting ligands. The siRNA molecule can be conjugated to the conjugation group non-covalently or covalently, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the siRNA to the conjugation group can be at the 3'-end or 5'-end of the sense strand or antisense strand of the siRNA, or can also be in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3'-end or 5'-end of the sense strand of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3'-end or 5'-end of the antisense strand of the siRNA. In some preferred embodiments, the conjugation site of the siRNA to the conjugation group is at the 3'-end of the sense strand of the siRNA.

[0206] 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 useful for targeting the liver in vivo for oligonucleotides and other molecules are known in the art. Galactose derivatives have been used to target molecules to hepatocytes in vivo through their binding to the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes. Binding of the ASGPR ligand to the ASGPR(s) facilitates cell-specific targeting of hepatocytes and endocytosis of the molecule into hepatocytes. The ASGPR ligand can be monomeric (e.g., having a single galactose derivative) or polymeric (e.g., having multiple galactose derivatives). Methods known in the art can be used to link galactose derivatives or clusters of galactose derivatives to the 3' or 5' end of the siRNA.

[0207] 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 molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule being 1:1, 1:2, 1:3, or 1:4, respectively, after the siRNA molecule forms an siRNA conjugate with the conjugating group containing the galactose or N-acetylgalactosamine molecule as the targeting ligand. In some embodiments, the pharmaceutically acceptable targeting ligand is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated with a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in the present disclosure is conjugated with a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0208] In some embodiments of the present disclosure, the targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid.

[0209] In some embodiments of the present disclosure, the targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

[0210] 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.

[0211] In some embodiments of the present disclosure, the targeting ligand is L96;

[0212] L96

[0213]

[0214] In some embodiments of the method of the present disclosure, the expression of the complement component C3 gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or is below the detection level. In a preferred embodiment, the expression of the complement component C3 is inhibited by at least 70%. It should also be understood that it may be desirable to inhibit the expression of the complement component C3 in certain tissues (e.g., the liver) without significantly inhibiting the expression in other tissues (e.g., the brain). In a preferred embodiment, the expression level is determined in a suitable species-matched cell line using the assay method provided in Example 2 at 50 nM, 10 nM and 1 nM siRNA concentrations.

[0215] In certain embodiments, the inhibition of in vivo expression is determined by knocking down the human gene in a rodent expressing the human gene, e.g., an AAV-infected mouse expressing the human target gene (i.e., the complement component C3), e.g., the lowest point of C3 expression after subcutaneous injection at 3 mg / kg as a single dose is used 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 such that human RNAi provides effective knockdown of the model animal gene. The RNA expression in the liver is determined using the PCR method provided in Example 2.

[0216] 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 in a sample derived from a subject, for example), in which the complement component C3 gene is transcribed in a cell or cell population and which is treated (e.g., by contacting one or more cells with an RNAi of the present disclosure, or by administering an RNAi of the present disclosure to a subject in which the cells are or were present), such that the expression of the complement component C3 gene is inhibited compared to a cell line that is substantially the same as the cell line but not treated (control cells not treated with RNAi or not treated with an RNAi targeting the target gene). In a preferred embodiment, the inhibition is evaluated using a 10 nM siRNA concentration in a species-matched cell line by the method provided in Example 2, and is represented by 2^-△△CT of the mRNA expression level in the treated cells relative to the mRNA level in the control cells using the following formula:

[0217] △CT = CT C3 - CT GAPDH

[0218] △△CT = △CT 处理细胞 - △CT 对照细胞

[0219] mRNA level = 2^-△△CT

[0220] In other embodiments, inhibition of complement component C3 gene expression can be evaluated based on a decrease in a parameter related to the function of complement component C3 gene expression, e.g., the complement component C3 protein level in the 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.

[0221] Inhibition of complement component C3 protein expression can be reflected by a decrease in the complement component C3 protein level or the secreted luciferase level expressed by a cell or cell population or in a subject sample (e.g., the protein level in a blood sample from a subject). As described above, for evaluating mRNA inhibition, the inhibition of the protein expression level in the treated cell or cell population can similarly be represented as a percentage of the protein level in the control cell or cell population, or the change in the protein level in a subject sample (e.g., blood or serum therefrom), and the inhibition is evaluated by the method provided in Example 3 or Example 4, using the following formula, and is represented by the percentage of the C3 expression level or Gluc expression level in the treated sample (e.g., blood or serum therefrom) relative to the C3 expression level or Gluc expression level in the control cells.

[0222] Percentage of mRNA inhibition = (protein expression level处理细胞 - Protein expression level 对照细胞 ) / Protein expression level 对照细胞 * 100%

[0223] Control cells, cell populations, or subject samples useful for assessing 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, prior to treating a subject or a properly matched group control with an RNAi agent, the control cells, cell lines, or subject samples can be from an individual subject (e.g., a human or animal subject).

[0224] In some embodiments of the methods of the present disclosure, the RNAi is administered to a subject such that the RNAi is delivered to a specific site within the subject. Inhibition of complement component C3 expression can be evaluated by measuring the level or change of complement component C3 mRNA or complement component C3 protein or fusion secreted luciferase in a sample of fluid or tissue from a specific site (e.g., liver or blood) of the subject.

[0225] The present disclosure also provides methods of using the RNAi of the present disclosure or a composition comprising the RNAi of the present disclosure to inhibit the expression of complement component C3, thereby preventing or treating complement component C3-related disorders, such as 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.

[0226] Cells suitable for treatment using the methods of the present disclosure can be any cells that express the complement component C3 gene, such as hepatocytes, brain cells, gallbladder cells, heart cells, or kidney cells, but preferably hepatocytes. Cells suitable for use in the methods of the present disclosure can be mammalian cells, such as primate cells (e.g., human cells, including human cells in chimeric non-human animals, or non-human primate cells, such as monkey cells or chimpanzee cells) or non-primate cells. In certain embodiments, the cells are human cells, such as human hepatocytes. 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 below the detected level of measurement.

[0227] The in vivo methods of the present disclosure can include administering to a subject a composition comprising RNAi, wherein the RNAi comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of the complement component C3 gene of the mammal to which the RNAi agent is administered. 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, parenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, 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 subcutaneously. In certain embodiments, the composition is administered by intramuscular injection.

[0228] In one aspect, the present disclosure also provides a method for inhibiting the expression of the complement component C3 gene in a mammal. The method includes administering to a mammal an oligonucleotide or a pharmaceutically acceptable salt thereof, a conjugate thereof, or a salt or composition thereof. The oligonucleotide is double-stranded RNA (dsRNA), and the dsRNA targets the complement component C3 gene in mammalian cells and is maintained in 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 cells. The reduction in gene expression can be evaluated by any method known in the art and by methods such as, for example, qRT-PCR described herein, for example, in Example 2. The reduction in the protein product can be evaluated by any method known in the art (e.g., ELISA). In other embodiments, a blood sample is used as the subject sample for monitoring the reduction in the expression of the complement component C3 protein.

[0229] The present disclosure also provides a method for treating in a subject in need thereof, e.g., a subject diagnosed with a complement component C3-related disorder, such as 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.

[0230] 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 causes clinical symptoms (e.g., livedo reticularis or acrocyanosis) associated with red blood cell (RBC) agglutination in cold parts of the body and hemolytic anemia. Cold agglutinins are IgM antibodies that recognize antigens on red blood cells (RBCs) at temperatures below normal core body temperature. They can cause RBC agglutination, complement activation, and extravascular hemolysis, resulting in anemia, usually without hemoglobinuria. CAD can be primary CAD (also known as idiopathic CAD) or secondary CAD. In subjects with primary CAD, cold agglutinins cause RBC agglutination and extravascular hemolysis without an underlying disease. In subjects with secondary CAD (also known as cold agglutinin syndrome, or CAS), cold agglutinins occur in the context of an underlying disease, such as viral infections, autoimmune diseases, or lymphoid malignancies (see, e.g., Berentsen (2015) Transfus Med Hemother 42:303-310).

[0231] 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 in body parts at a temperature equal to or higher than normal body temperature, and hemolytic anemia results from the activation of the complement system by IgG antibodies directed against blood group antigens. 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 the cases of warm autoimmune hemolytic anemia are primary because no specific cause can be found, while the remaining cases are thought to be secondary to lymphoproliferative syndromes; malignant diseases, including chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, and solid tumors; rheumatic diseases, particularly systemic lupus erythematosus; infections (mainly viral); drugs; frequent use of cephalosporins and piperacillin; or prior blood transfusion or transplantation (see, e.g., Berentsen (2015) Transfus Med Hemother 42:303-310).

[0232] In one embodiment, the complement component C3-related disease is paroxysmal nocturnal hemoglobinuria (PNH). PNH can be classical 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, and is characterized by complement-mediated hemolytic anemia, a tendency to thrombosis, and bone marrow failure (see, e.g., Risitano (2013) Adv Exp Med Biol 735:155).

[0233] In one embodiment, the complement component C3-related disease is lupus nephritis (LN), i.e., any one of classes I to VI lupus nephritis. LN is a glomerulonephritis caused by systemic lupus erythematosus (SLE). The occurrence of lupus nephritis is due to the deposition of immune complexes in any or all renal compartments, including glomeruli, tubules, and interstitium. IgG is the most common antibody, but IgM and IgA can also be seen. These autoantibodies lead to the activation of the classical and alternative complement pathways, and thus C1, C3, and properdin can be detected in biopsies.

[0234] In one embodiment, the complement component C3-related disease is bullous pemphigoid. Bullous pemphigoid is an autoimmune blistering disease caused by autoantibodies against type XVII collagen (COL17), which can activate complement and subsequently recruit inflammatory cells at the dermal / epidermal junction. Bullous pemphigoid is the most common autoimmune blistering disorder, characterized by tense blisters accompanied by itchy urticarial erythema and plaques throughout the body.

[0235] In one embodiment, the complement component C3-related disease is pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Pemphigus is a group of rare chronic blistering diseases characterized by IgG autoantibodies against multiple desmosomal transmembrane glycoproteins and intracellular deposition of IgG and C3c. Patients with pemphigus vulgaris typically present with oral mucosal lesions followed by skin involvement, and the autoantibodies are directed against the epithelial adhesion proteins desmoglein 3 and / or desmoglein 1. In pemphigus foliaceus, the lesions are limited to the skin and do not involve the mucosa, and the autoantibodies are directed against desmoglein 1. In one embodiment, the pemphigus is pemphigus vulgaris (PV). In another embodiment, the pemphigus is pemphigus foliaceus (PF).

[0236] In one embodiment, the complement component C3-related disease is C3 glomerulopathy. C3 glomerulopathy is characterized by the activation of the alternative complement cascade and the deposition of complement component C3, without any immunoglobulin deposition in the glomeruli of the kidney.

[0237] The RNAi of the present disclosure can be administered as "naked RNAi". Naked RNAi is administered without a pharmaceutical composition. The naked RNAi can be in a suitable buffer solution. The buffer solution can comprise 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 the RNAi can be adjusted to be suitable for administration to a subject.

[0238] Administration of RNAi according to the methods of the present disclosure can result in the prevention or treatment of complement component C3-related disorders, such as, 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 therapeutically effective amount of RNAi, such as from about 0.01 mg / kg to about 200 mg / kg, can be administered to a subject. Preferably from 1 mg / kg to about 50 mg / kg. The RNAi is preferably administered subcutaneously, i.e., by subcutaneous injection. One or more injections can be used to deliver the desired dose of RNAi to the subject. The injections can be repeated over a period of time.

[0239] In some embodiments, the RNAi is administered at a fixed dose of from about 10 mg to about 800 mg. In some embodiments, the RNAi is administered to a subject at a fixed dose of from about 10 mg to 50 mg, from about 50 mg to about 200 mg, from about 200 mg to about 400 mg, or from about 400 mg to about 800 mg. In some embodiments, the 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.

[0240] The administration can be repeated regularly. In certain embodiments, after an initial treatment regimen, treatment can be performed at a lower frequency. The repeated dosing regimen can include regular administration of a therapeutically effective amount of RNAi, such as from once a month to once a year. In certain embodiments, the 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.

[0241] The present disclosure further provides for the combined treatment of a subject who would benefit from a reduction and / or inhibition of C3 gene expression, e.g., a subject suffering from a C3-related disease, with an RNAi agent or a pharmaceutical composition thereof in combination with other drugs and / or other therapeutic methods (e.g., known drugs and / or known therapeutic methods, such as, for example, those currently used to treat these conditions). For example, other therapeutic agents and therapeutic methods suitable for treating a subject who would benefit from a reduction in C3 expression (e.g., a subject suffering from 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 the signaling of pro-inflammatory cytokines (such as 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).

[0242] Examples

[0243] Example 1. Preparation of Targeting Ligand and siRNA

[0244] In the case where the source of the reagent is not specifically given herein, such reagents can be obtained from any molecular biology reagent supplier, and their quality / purity standards are applicable to molecular biology.

[0245] Abbreviations of nucleotide monomers used in nucleic acid sequence representation.

[0246] Table A. Abbreviations of nucleotide monomers used in nucleic acid sequence representation

[0247]

[0248]

[0249] Preparation of Targeting Ligand

[0250] The preparation of L96 was carried out according to the method described in Patent CN104717982B.

[0251] Preparation of Oligonucleotide

[0252] (1) Preparation of siRNA

[0253] First, computer-based algorithms were used to generate candidate oligonucleotide sequences complementary to human C3 mRNA (NM_000064.3, Table 1), some of which were also complementary to cynomolgus monkey C3 mRNA (XM_005587719.3, Table 1) or had no more than 2 mismatches. Some of them were designed as double-stranded siRNAs with 19 / 21 pairing of the sense and antisense strands respectively, and the antisense strand had two overhangs complementary to the mRNA sequence. In some cases, the overhangs of the antisense strand were non-complementary UU; some of the sequences were designed as double-stranded siRNAs with 21 / 23 pairing of the sense and antisense strands respectively, and the antisense strand had two overhangs complementary to the mRNA sequence; some of the sequences were designed as double-stranded siRNAs with 21 / 21 or 23 / 23 pairing. For some of the complementary pairing sequences, the base at the 1st position at the 5' end of the antisense strand (the last position at the 3' end of the sense strand) was replaced with a base that did not match the C3 mRNA.

[0254] Table 1 Human and cynomolgus monkey C3 mRNA sequences

[0255] species GenBank RefSeq# human NM_000064.3 cynomolgus monkey XM_005587719.3

[0256] The siRNA sequences were synthesized separately on a solid support via the sense strand (SS) and antisense strand (AS), and obtained after deprotection, cleavage, purification, annealing, purification and lyophilization.

[0257] Solid-phase synthesis ( Figure 2):The sense strand and the antisense strand are respectively synthesized on a solid support using the phosphoramidite technology with an oligonucleotide synthesizer. The synthesizer is such as AKTA Oligopilot (Cytiva), Dr. Oligo 192XLc (Kunshan Berlick Precision Instruments Co., Ltd.). Solid-phase synthesis starts from the 3'-end of the sequence, and monomers are coupled into the sequence in sequence order. For each coupling of a phosphoramidite monomer, it includes four chemical steps: 1) deblocking or deprotection (removing the hydroxyl protecting group); 2) coupling; 3) oxidation; 4) capping. The phosphoramidite monomers, reagents, and purification consumables used are all commercially available reagents and consumables. For example, various phosphoramidite monomers (such as 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-Uridine-3'-CE-Phosphoramidite) are purchased from Shanghai Zhaowei Technology Development Co., Ltd., and reaction reagents (such as 40wt% aqueous methylamine solution, 28wt% aqueous ammonium hydroxide solution, etc.) are purchased from Sigma-Aldrich LLC. The siRNA synthesis and purification methods used in this article are as described in US20130178612A1, US2015100197A1, etc.; the synthesis method of the sequence containing VPUm and APU structures is as described in J. Med. Chem. 2018, 61, 734 - 744.

[0258] (2) Preparation of double-stranded RNA reagent

[0259] (a) Synthesis of sense strand

[0260] The solid-phase phosphoramidite method is a well-established method for oligonucleic acid synthesis. The reaction is carried out in a stainless-steel synthesis column using a computer-controlled synthesizer. In the synthesis of the sense strand, starting from a solid support loaded with a targeting ligand (such as L96), or directly starting from a solid support, different raw materials, reagents, and solvents are injected in sequence from 3' to 5' of the sequence through different pipelines of the solid-phase synthesizer, and phosphoramidite nucleoside monomers are connected one by one. The reaction process includes four-step cycles of DMT protecting group removal reaction, condensation reaction, oxidation or thiolation reaction, and capping reaction. Each cycle connects one nucleotide unit to obtain an oligonucleic acid sequence of 19 or 21 nucleotide units. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and then the synthesized sequence is cleaved from the solid support through an ammonolysis reaction, filtered, the filter cake is washed with ethanol, and the filtrate and washing solution are collected and concentrated to obtain the crude sense strand. The crude product is purified by chromatography (SOURCE 15Q) and lyophilized to obtain the target product, the sense strand. Among them, in the synthesizer, starting from a solid support loaded with a targeting ligand (such as L96), the siRNA sense strand conjugate was synthesized; starting directly from a solid support, siRNA was synthesized.

[0261] (b) Synthesis of Antisense Strand

[0262] The synthesis of the antisense strand is similar to that of the sense strand. Using a solid-phase synthesizer, different raw materials, reagents, and solvents are injected through different pipelines in the order from 3' to 5' of the sequence to sequentially link phosphoramidite nucleoside monomers. The reaction process includes four-step cycles of DMT protecting group removal reaction, condensation reaction, oxidation or thiolation reaction, and capping reaction. Each cycle attaches one nucleotide unit to obtain an oligonucleotide sequence of 21 or 23 nucleotide units. After synthesis, the protecting group (2-cyanoethyl) is removed on the solid-phase synthesis column, and then the synthesized sequence is cleaved from the solid support through an ammonolysis reaction, filtered, the filter cake is washed with ethanol, and the filtrate and washing solution are collected and concentrated to obtain the crude antisense strand. The crude product is purified by chromatography (SOURCE 15Q), ultrafiltered, and lyophilized to obtain the target product, antisense strand siRNA.

[0263] (c) Preparation of Double-Stranded siRNA

[0264] The AS strand and the SS strand are separately dissolved in injection water, mixed in a determined ratio (1.01:1.0 - 1.2:1.0), incubated at 30 - 50 °C for 30 - 90 min, and then cooled to room temperature. After freeze-drying, the double-stranded siRNA product is obtained.

[0265] According to the same method, the double-stranded siRNA agents in Tables 2, 3, and 4 below are prepared.

[0266] In Tables 2, 3, and 4, "G", "C", "A", "U", "T", and "I" generally represent nucleotides with guanine, cytosine, adenine, uracil, thymine, and hypoxanthine as bases respectively. The naked sequences in Tables 2, 3, and 4 refer to unmodified oligonucleotide sequences.

[0267] Modifications: m represents 2'-methoxy; f represents 2'-deoxy-2'-fluoro; s represents phosphorothioate; VPUm is 2'-methoxy-modified uridine; M form is 2'-O-methyl-6-(3-(2-carboxyethyl)phenyl)-purine nucleotide; L96 is N-[tris(GalNAc-alkyl)amido decanoyl]-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.

[0268] VPUm:

[0269]

[0270] M:

[0271]

[0272] L96:

[0273]

[0274] Table 2 Naked Oligonucleotide Sequences

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] Table 3 Modified Oligonucleotide Sequences

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] Table 4 siRNA Sequences with Targeting Ligands

[0292]

[0293]

[0294] Example 2 In Vitro Activity Screening of C3-siRNA Naked Sequences

[0295] (1) Cell Culture and Transfection:

[0296] Human liver cancer cells (undifferentiated) (also known as HLE cells) (Wuhan Pusai Biotechnology Co., Ltd., product number CL-0651) and Hep3B cells (Wuhan Pusai Biotechnology Co., Ltd., product number CL-0102) were placed in an incubator at 37°C and 5% CO2, and cultured using DMEM medium (Saibakang (Shanghai) Biotechnology Co., Ltd., product number 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), counted using a cell counter (Countstar, IC1000), and 190 μl of cell suspension per well was seeded into a 96-well plate. The seeding number of HLE cells was: 5*10 4 cells / well, and the seeding number of Hep3B cells was 2*10 4 cells / well, and they were allowed to adhere overnight before transfection.

[0297] Transfection was performed using LipofectamineTM RNAiMAX (Thermo Fisher, 13778150). A transfection complex was prepared by mixing 2.2 μl (2 μM) of diluted compound, 19.1 μl of Opti-MEM (Thermo Fisher, 1105821), and 0.7 μl of RNAiMAX. After incubation for 5 minutes, the transfection complex was added to the cells (two technical replicates for each complex), 10 μl per well, and the final concentration of siRNA was 10 nM. The cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0298] (2) RNA extraction and detection

[0299] (i) Total RNA was extracted using the RNA-Quick Purification Kit (RNA fast extraction kit, Yishan Bio, RN001):

[0300] Take out the 12-well plate from the incubator, suck out the culture medium, wash it once with an appropriate amount of PBS, add 500 μl of lysis buffer to each well, and transfer the supernatant to a new 1.5-ml centrifuge tube. Add 500 μl of absolute ethanol to the lysed cells and mix well (if precipitation occurs, this is normal and the operation can continue). Invert the centrifuge tube several times or pipette and blow 10 times forcefully to disperse the generated precipitate, then add the liquid to the centrifugal column. Place the centrifuge tube symmetrically in the centrifuge (eppendorf, 5430) and centrifuge at 4000×g for 1 min. Take out the centrifuge tube, add 500 μl of washing buffer to the column, centrifuge at 12000×g for 1 min. After centrifugation, take out the column, pour out the waste liquid, reinstall the RNA column into the collection tube, and centrifuge the empty tube once to remove the possibly remaining washing buffer. Place the column on a clean RNase-free 1.5-ml centrifuge tube, open the lid and air-dry for 2 minutes. Add 30 μl of elution buffer to the center of the membrane of the RNA column, let it stand at room temperature for 2 minutes, centrifuge at 2000×g for 1 min. After the RNA is eluted, place it on ice. Measure the concentration of the eluted RNA for subsequent experimental use. The extracted RNA can be immediately used for subsequent experiments or stored at -80 °C for later use.

[0301] (ii) Synthesize cDNA using IIQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme, R223-01):

[0302] Prepare a mixture in a RNase-free centrifuge tube: 4 μl of 4×gDNA wiper Mix, 1 μg of template RNA, add RNase-free ddH2O to 16 μl to remove genomic DNA, gently pipette and mix well, incubate at 42 °C for 2 min. Then directly add 4 μl of 5×HiScript II qRT SuperMix II to the reaction tube, gently pipette and mix well, place it in the PCR instrument (Applied Biosystems, 9700) at 50 °C for 15 min; 85 °C for 5 sec, hold at 4 °C. The product can be immediately used for qPCR reaction or stored at -20 °C and used within half a year. For long-term storage, it needs to be aliquoted and stored at -80 °C. cDNA should be avoided from repeated freezing and thawing.

[0303] (iii) Perform qPCR quantification using ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02):

[0304] Prepare a 20 μl reaction system by adding 10 μl of 2× ChamQ SYBR qPCR Master Mix, 0.5 μl of Forward primer (Ruibo Xingke), 0.5 μl of Reverse primer (Ruibo Xingke), 1 μl of Template cDNA, and 8 μl of ddH2O. Each sample has 3 replicates. Place the 96-well plate in a qPCR instrument (ROCGENE, Archimed) and execute the following program: pre-denaturation at 95 °C for 30 sec; amplification at 95 °C for 10 sec, 60 °C for 30 sec, for 40 cycles; melting curve at 95 °C for 15 sec, 60 °C for 60 sec, 95 °C for 15 sec.

[0305] (3) Data statistical analysis:

[0306] Export the data in EXCEL format and use CT C3 -CT GAPDH . Normalize the control group. To calculate the fold change of relative silencing efficiency, the data is analyzed using the ΔΔCT method. Calculate the average value and standard deviation of the three parallel replicate data obtained.

[0307] The screening results of HLE cells for 2 times are shown in Table 5 - Table 6.

[0308] As shown in Table 5, when the drug dosage is 50 nM, the inhibition rate of C3 mRNA by 56 siRNAs can reach over 70%, and the inhibition rate of some sequences on C3 mRNA can reach over 80%, even over 90%. To further evaluate more optimal sequences, 36 sequences with better inhibition rates were selected for double-concentration screening at drug dosages of 10 nM and 1 nM. The detection results are shown in Table 6. It can be seen that when the drug dosage is further decreased, the double-concentration screening results of AL0161002, AL0161005, AL0161006, AL0161015, AL0161018, AL0161020, AL0161037, AL0161041, AL0161048, AL0161067, and AL0161091 are relatively optimal, with the inhibition rate at 10 nM reaching over 60% or the inhibition rate at 1 nM also approaching 60%.

[0309] Table 5 Knockdown levels of C3 siRNA naked sequences in HLE cells

[0310]

[0311]

[0312]

[0313] Table 6 Knockdown level of naked C3 siRNA sequence in Hep3B

[0314]

[0315]

[0316] Example 3. In vitro activity screening of chemically modified C3-siRNA

[0317] (1) Cell culture and transfection:

[0318] (i) The culture and transfection of Hep3B cells were the same as in Example 2.

[0319] (ii) Cultivation and transfection of cynomolgus monkey hepatocytes

[0320] Use cynomolgus monkey hepatocytes (Beijing Red Biotech Co., Ltd., cmTCSC). Preheat the culture medium first, take out the thawing culture medium (Beijing Red Biotech Co., Ltd., HEPO24) to the biosafety cabinet, add 4 mL of FBS to 36 ml of thawing culture medium (TPCS, HEPO24) to make a complete thawing culture medium, and heat it in a 37°C water bath for 10 minutes. Treat with coating medium (Beijing Red Biotech Co., Ltd., HEPO44) in a CO2 incubator at 37°C for 0.5 h. Take out the cells from liquid nitrogen, revive the cells in a 37°C water bath, take them out after about 2 minutes, transfer the cell suspension to 40 ml of preheated thawing culture medium, wash the cell cryopreservation tube with 2 ml of complete thawing culture medium, centrifuge the cell suspension at 180×g for 1 minute, discard the supernatant, add 2 ml of preheated CM seeding culture medium (Beijing Red Biotech Co., Ltd., CMHEP054), gently blow the cell suspension to mix, and take 20 μl of cell suspension for counting. According to the counting results, 12-well plates were inoculated with 3*10 5 / well, and cultured in an incubator at 37°C and 5% CO2. After 4-5 hours of adhesion, the CM seeding medium was aspirated and replaced with pre-warmed medium (Beijing Red Biotech Co., Ltd., CMHEP064), and transfection was performed after 6 hours of adhesion.

[0321] Use of Lipofectamine TMTransfect with 3000 Transfection Reagent (Thermo Fisher, L3000150). For System ①, dilute 50 nM modified siRNA (Suzhou Beixin Biotechnology Co., Ltd.) with 50 μl of Opti-MEM (Thermo Fisher, 1105821). For System ②, dilute 3 μl of Lipo3000 with 50 μl of Opti-MEM. Let them stand for 5 min respectively, then mix System ① and ② and let it stand for another 15 min. Drop the mixture into a 12-well plate. After 4 h of transfection, replace it with DMEM / F12 complete medium, and incubate the 12-well plate in an incubator for 48 h.

[0322] (2) RNA extraction and detection

[0323] Same as Example 2.

[0324] (3) Data statistical analysis:

[0325] Same as Example 2.

[0326] As can be seen from Table 7, in primary crab-eating macaque hepatocytes, when the dosage is 1 nM, except for AL0165002 and AL0165008, the inhibition rates of all sequences on C3 mRNA can reach 60%. In particular, the inhibition rates of sequences AL0165005, AL0165006, AL0165009, AL0165010, and AL0165011 on C3 mRNA can reach 70%, even close to 80%.

[0327] As can be seen from Table 8, in Hep3B cells, when the dosage is 10 nM, the inhibition rates of 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 on C3 mRNA can be greater than 80%, even close to 90%, and when the dosage is 1 nM, it can reach at least 60% on C3 mRNA. After partial base mismatches or adding VPUm modifications to the sequences with better efficacy, the efficacy can be further improved, as shown in Table 9 and Table 10. Further test the sequences with even better efficacy in primary crab-eating macaque hepatocytes. Each batch of experiments is compared with AL0165001 to screen out the sequences with better efficacy. As shown in Table 11 - Table 13, AL0165048, AL0165088, AL0165112, AL0165116, AL0165124, AL0165129, AL0165136, AL0165146, AL0165167, AL0165171, AL0165177, AL0165178, AL0165188, AL0165189, AL0165191, AL0165192, AL0165193, AL0165195, AL0165205, AL0165207, AL0165210, AL0165221, AL0165223, AL0165225, AL0165227, AL0165229, AL0165231, AL0165232, AL0165233, AL0165234, AL0165235, AL0165236, AL0165237 and AL0165238 perform better.

[0328] In another in vitro activity screening experiment of Hep3B, as can be seen from Table 14, when the dosage is 1 nM and 0.1 nM, the efficacy of sequences with different modifications can be further improved. For example, the inhibition rates of AL0165242, AL0165217, AL0165246, AL0165218, AL0165247, and AL0165248 on C3 mRNA can exceed 85%, and even exceed 90%. Especially when the antisense strand contains a 2'-5'-phosphodiester bond or a substitution modification at some positions with Im, the efficacy can be improved.

[0329] In another screening experiment of in vitro activity in cynomolgus monkey primary hepatocytes, as shown in Table 15, when the dosage is 1 nM and 0.1 nM, the efficacy of sequences with different modifications can be further improved. For example, the inhibition rates of AL0165242, AL0165217, AL0165246, AL0165218, AL0165247, and AL0165248 on C3 mRNA can exceed 85%, and even exceed 90%. Especially when the antisense strand contains a modification with a 2'-5'-phosphodiester bond or a substitution modification at some positions with Im, the efficacy can be improved.

[0330] Table 7 Knockdown levels of chemically modified C3 siRNA in cynomolgus monkey primary hepatocytes

[0331]

[0332] Table 8 Knockdown levels of chemically modified C3-siRNA in Hep3B

[0333]

[0334]

[0335]

[0336]

[0337] Table 9 Knockdown levels of chemically modified C3-siRNA in Hep3B

[0338]

[0339]

[0340] Table 10 Knockdown levels of chemically modified C3-siRNA in Hep3B

[0341]

[0342] Table 11 Knockdown levels of chemically modified C3 siRNA in cynomolgus monkey primary hepatocytes

[0343]

[0344]

[0345] Table 12 Knockdown levels of chemically modified C3 siRNA in primary crab-eating macaque hepatocytes

[0346]

[0347] Table 13 Knockdown levels of chemically modified C3 siRNA in primary crab-eating macaque hepatocytes

[0348]

[0349] Table 14 Knockdown levels of chemically modified C3 siRNA in Hep3B cells

[0350]

[0351]

[0352] Table 15 Knockdown levels of chemically modified C3 siRNA in primary monkey hepatocytes

[0353]

[0354] Example 4. In vivo testing of C3 RNAi agent in AAV transgenic mice of C3

[0355] Gluc is easily secreted and highly sensitive, and can directly measure the expression activity of Gluc in whole blood. Gluc is linked to foreign genes through P2A, and the expression level of Gluc directly reflects the mRNA level of foreign genes. The mRNA sequence of the human C3 gene (NM_000064.3) was obtained from the NCBI database, and recombinant plasmids were obtained through conventional molecular biology techniques such as enzymatic digestion and ligation. The 93-2893 fragment (HsC3_P1) and 2293-4531 fragment (HsC3_P2) in the C3 mRNA sequence were secreted and expressed in the form of fusion proteins with Gaussia luciferase (Gluc) (the gene sequences of the fusion proteins are 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, catalog 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 through conventional molecular biology techniques such as enzymatic digestion and ligation to obtain RA177 pFB-AAV-CAG-Gluc-2A-HsC3_P1( Figure 3 ) and RA178 pFB-AAV-CAG-Gluc-2A-HsC3_P2( Figure 4 ) vectors. The two plasmids were transfected into Sf9 cells (ATCC CRL-1711 TM ) respectively to obtain AAV viruses 146-177 and 146-178 containing the hC3 gene, and then administered by tail vein injection at least 29 days before administering the C3 RNAi reagent or control.

[0356] SPF-grade male C57BL / 6 mice, 6-8 weeks old (Spearf Bio (Beijing) Biotechnology Co., Ltd.) were used in the experiment. Each mouse was injected with 1.00E+12 vg / mL of 146-177 or 146-178 AAV virus particles via the tail vein. After 14 days of injection, blood was collected from the orbital cavity, and the serum was separated for Gluc luminescence detection (Pierce TMGaussia luciferase glow detection kit, Thermo, 16160), randomly divided into two vehicle control groups (NC group) according to the C3-Gluc luminescence detection value, and the test substances AL0167001, AL0167002, AL0167003, AL0167004, AL0167005, AL0167006, AL0167007, AL0167008, AL0167009, AL0167010, AL0167011, AL0167012, AL0167013, AL0167014 and AL0167015, a total of 15 groups. Among them, Figure 5 In A, AL0167001, AL0167002, AL0167003, AL0167004, AL0167005, AL0167006, AL0167007, AL0167008, AL0167009, AL0167012 and AL0167013 were injected with 146-177 AAV virus; Figure 5 In B, the AL0167001, AL0167008, AL0167009, AL0167010, AL0167011, AL0167014 and AL0167015 groups were injected with 146-178 AAV virus, a total of 17 groups, with 5 mice in each group. After grouping, the mice were subcutaneously administered the corresponding C3 RNAi agent or vehicle control once. Blood was collected from the mice before dosing and on days 8, 15 and 22 after dosing, and the serum was separated for luc luminescence detection to evaluate the inhibitory effect of siRNA on exogenous gene mRNA. The results are shown in Figure 5 . Among them, the groups with the most significant inhibitory effect on C3 were the AL0167002, AL0167006, AL0167009, AL0167013 and AL0167015 groups.

[0357] Example 5. In vivo test of C3 RNAi agent in transgenic mice

[0358] The experiment used SPF-grade male hC3, NM-HU-2000079 mice at 8-9 weeks of age (purchased from Model Animal Research Center). Serum samples were obtained before dosing on day 0, and the mice were randomly grouped according to the hC3 level. The hC3, NM-HU-2000079 mice were subcutaneously administered 3 mg / kg of C3 RNAi agents AL0167001, AL0167006, AL0167009, AL0167013, and AL0167015 once. Blood samples were collected from the mice at 1 week, 2 weeks, 3 weeks, 4 weeks, and 5 weeks after dosing respectively (by eye bleeding and sent for inspection within 1 h after blood collection), and the hC3 expression level was detected. Taking the pre-dose as the control, the knockdown level of C3 was detected. During the experiment, no death or near-death symptoms were observed in all animals. No obvious abnormalities were observed in all animals during clinical observation. The change level of hC3 is shown in Figure 6 A.

[0359] In addition, for the in vivo activity test of the other two groups of transgenic mice, the hC3, NM-HU-2000079 mice were subcutaneously administered 3 mg / kg of C3 RNAi agents once. Among them, one group of drugs was AL0167016, AL0167017, AL0167018, AL0167019, AL0167020, AL0167021, and AL0167022, and the other group of drugs was AL0167023, AL0167024, AL0167025, and AL0167026. Blood samples were collected from the mice at 1 week, 2 weeks, 3 weeks, 4 weeks, and 5 weeks after dosing respectively for the two groups of experiments (by eye bleeding and sent for inspection within 1 h after blood collection), and the hC3 expression level was detected. Taking the pre-dose as the control, the knockdown level of C3 was detected. During the experiment, no death or near-death symptoms were observed in all animals. No obvious abnormalities were observed in all animals during clinical observation. The change level of hC3 is shown in Figure 6 B and 6C.

[0360] It can be concluded that Figure 6 compared with before dosing, the knockdown effect of all C3 siRNAs reached the lowest on the 7th day after drug intervention, gradually and slowly rebounded on the 14th day, and the level of hC3 in the blood was significantly knocked down during the 35-day detection period. No drug-related deaths occurred in all test groups, and drug intervention could significantly reduce the hC3 level in the blood of mice. Compared with the vehicle control group (NC group), all test groups had a 50%-70% knockdown to varying degrees.

Claims

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

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 carboxylate, alkali metal salt, ammonium salt, alkaline earth metal salt, salt formed with an organic base 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 or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: The oligonucleotide comprises at least one modified nucleotide; Preferably, the oligonucleotide comprises at least one 2'-modified nucleotide; Preferably, the 2'-modified nucleotide is 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 analog modified nucleotide at the 5' end; preferably, the 5'-phosphate analog 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 analog 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 analog 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 shown in formula (V); Preferably, the oligonucleotide comprises a uridine-2'-phosphate (U-2'5') selected from the group consisting of uridine-2'-phosphate (U-2'5') represented by formula (VI), guanosine-2'-phosphate (G-2'5') represented by formula (VII); cytidine-2'-phosphate (C-2'5') represented by formula (VIII); adenosine-2'-phosphate (A-2'5') represented by formula (IX) and thymidine-2'-phosphate (T-2'5') represented by formula (X); 4. The oligonucleotide or a 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 bond is a phosphorothioate bond.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: The sense strand is selected from the unmodified oligonucleotides described in 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 oligonucleotides described in any one of SEQ ID NO.588-791; the antisense strand is selected from the unmodified oligonucleotides described in any one of SEQ ID NO. 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 of any unmodified oligonucleotide, or SEQ ID The modified oligonucleotide described in any one of NO.793-1001, 1010-1021.

6. The oligonucleotide or a 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 a 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) an 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 or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 or the conjugate or a pharmaceutically acceptable salt thereof according to claim 8, and optionally a pharmaceutically acceptable carrier; Preferably, the composition is in the form of an oral agent, 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 the treatment and / or prevention of 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.

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