Dsrna for inhibiting c5 gene expression and use thereof

Abstract

Provided are a double-stranded ribonucleic acid (dsRNA) for inhibiting C5 gene expression, a double-stranded RNAi agent, and a use thereof. The dsRNA and the double-stranded RNAi agent can be used for inhibiting C5 gene expression, thereby preventing and treating diseases and / or conditions related to C5 gene expression.

Description

A dsRNA for inhibiting C5 gene expression and its use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure is based on and claims the priority of Chinese patent application with application number 202410027758.8, application date January 8, 2024, and invention name “A dsRNA for inhibiting C5 gene expression and its use”. The entire content of the Chinese patent application is hereby incorporated into the present disclosure by reference. Technical Field

[0003] The present disclosure belongs to the field of biomedicine, and specifically relates to a double-stranded RNA for inhibiting C5 gene expression and its use, especially for preventing or treating diseases and / or conditions related to C5 gene expression. Background Art

[0004] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening blood disorder caused by a genetic mutation and characterized by complement-mediated intravascular hemolytic anemia, hemoglobinuria, hypercoagulability, and thrombosis.

[0005] PNH is the only known acquired hemolytic anemia. It is caused by an acquired (rather than inherited) genetic defect in the red blood cell membrane, resulting in a deficiency of glycophosphatidylinositol. This leads to a lack of protective proteins on the cell membrane, causing the complement system to mistakenly attack the body's own red blood cells, causing erythrocyte lysis and the release of hemoglobin, leading to hemolytic anemia. It can occur in isolation ("primary PNH") or in the context of other bone marrow disorders such as aplastic anemia ("secondary PNH"). Allogeneic bone marrow transplantation is currently the only curative treatment, but it carries a high mortality rate and ongoing morbidity.

[0006] The complement system is an ancient, evolutionarily conserved, nonspecific host defense system that assists and supplements specific antibody responses, mediating immune bacteriolysis and hemolysis, hence the name complement. Initially considered a defense against bacterial infection, subsequent research has shown that the complement system plays a crucial role in maintaining homeostasis, opsonizing apoptotic cell fragments, and initiating adaptive immune responses. Complement proteins comprise approximately 50 serum and membrane components, which form a cascade of serine proteases, ultimately forming the membrane attack complex (MAC), which lyses bacteria and cells. Although other tissues also release complement components locally, serum components of the complement system, such as C3 and C5, are primarily produced and secreted by the liver. The complement cascade is initiated through three pathways: the antibody-mediated classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). The AP pathway can also amplify the CP and LP pathways.

[0007] The CP pathway initiates with antibody binding and recognition by the C1q / r / s complex, which cleaves C2 and C4 to form the C3 convertase C2aC4b. The LP pathway initiates with recognition of cell surface lectins by mannose-binding lectin (MBL) and subsequent activation of MASP-1 and MASP-2. The MBL / MASP-1 / -2 complex also activates C2 and C4, forming the C3 convertase C2aC4b, identical to the classical pathway. The AP pathway can be activated by a variety of substances, including negatively charged surface substances, lipopolysaccharide (LPS), and C3(H2O) generated by spontaneous hydrolysis of C3. Activated factor D cleaves factor B-bound C3b to form the C3 convertase C3bBb. Both C3 convertases cleave C3 to form C3b and release the anaphylatoxin C3a. C3b binds to various convertases to form C5 convertase, which cleaves C5 into C5b, initiating the formation of the C5b-9 membrane attack complex. All three complement activation pathways ultimately converge on the cleavage and activation of C3 and C5, generating the anaphylatoxin C5a and C5b, which subsequently form the membrane attack complex (C5b-9). C5a exerts its proinflammatory activity by interacting with the G protein-coupled receptor C5aR (CD88) and the non-G protein-coupled receptor C5L2 (GPR77). C5b-9 induces cytolysis through the formation of the membrane attack complex (MAC), and soluble C5b-9 also has numerous non-cytolytic immune functions. C5a and C5b, generated from C5 cleavage, are key components in various diseases, including paroxysmal nocturnal hemoglobinuria, rheumatoid arthritis, ischemia-reperfusion injury, and neurodegenerative diseases.

[0008] To date, the only antibody drugs targeting the C5-C5a axis are Soliris (the anti-C5 antibody eculizumab) and its long-acting optimized product, Ultomiris (ravulizumab), which have been approved for the treatment of complement component C5-related diseases, including paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS).

[0009] The monoclonal antibody eculizumab (Soliris) has been shown to effectively reduce the need for blood transfusions, improve quality of life, and reduce the risk of thrombosis. Eculizumab specifically binds to terminal complement component 5 (C5), which plays a role in the later stages of the complement cascade. When activated, C5 participates in the activation of host cells, thereby attracting proinflammatory immune cells, while also destroying cells by triggering pore formation. By inhibiting the complement cascade at this time, the normal, disease-preventing function of the proximal complement system is largely preserved, while the proinflammatory and cell-destructive properties of C5 are hindered.

[0010] It is important to note that eculizumab therapy requires weekly high-dose infusions during the initial treatment phase, followed by biweekly infusions during the subsequent maintenance phase, at an annual cost of approximately $400,000. Even eculizumab's long-acting optimized product, revoluizumab, requires high-dose infusions every two weeks and then every eight weeks, at an annual cost of approximately $508,000. Therefore, there is an urgent need to develop the next generation of highly effective, long-acting alternative therapies and / or combination therapies for PNH patients in the field of complement activation.

[0011] Summary of the Invention

[0012] The present disclosure provides an inhibitor for inhibiting C5 gene expression, such as an RNAi agent or RNA, and a pharmaceutical composition thereof, and use thereof in preparing a medicament for preventing, treating, and / or inhibiting related diseases.

[0013] The RNAi agents and RNA disclosed herein are designed to target the C5 gene, including portions of the gene that are conserved in orthologs of other mammalian species. RNAi agents typically comprise a sense strand and an antisense strand that form a duplex, double-stranded RNA (referred to herein as "dsRNA"). RNAi agents comprising dsRNA are also referred to herein as "dsRNAi" agents.

[0014] Without intending to be limited by theory, the RNAi agents and RNAs disclosed herein, as well as specific target sites and / or modifications in these RNAi agents and RNAs, confer improved efficacy, stability, potency, durability, and / or safety. For example, but not limited to, in some embodiments, the RNAi agents and / or RNAs disclosed herein exhibit: (1) improved efficacy and / or potency, for example, by stronger hybridization with target gene mRNA; and / or, (2) improved safety, for example, by reducing off-target effects, for example, by reducing or attenuating hybridization with off-target RNAs.

[0015] The use of RNAi agents of the present invention can enable targeted degradation of the mRNA of the C5 target gene in mammals. The inventors have confirmed that the RNAi agents of the present invention can trigger the cleavage of C5 RNA transcripts mediated by RNA-induced silencing complex (RISC), thereby significantly inhibiting the expression of C5 target genes. In certain embodiments, the RNAi agents of the present invention are more effective (e.g., more potent) and / or more specific (e.g., safer, with fewer off-target effects) than previous RNAi agents targeting the same gene. In certain embodiments, RNAi agents are targeted to specific sites in C5 mRNA by selecting a specific site, and / or include RNA modifications (e.g., modified nucleotides, chemical modifications) to increase efficacy, effectiveness, specificity, and / or safety. In some such embodiments, the RNAi agent comprises at least one modified nucleotide. The methods and compositions comprising these RNAi agents can be used to treat subjects suffering from C5-related diseases or disorders, such as C5-related disorders. Therefore, the present disclosure provides methods for treating, preventing, or inhibiting C5-related disorders in subjects, who will benefit from the inhibition or reduction of C5 expression caused by the use of the RNAi agents and compositions of the present invention.

[0016] In one aspect, the present disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting C5 expression, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, the antisense strand comprises at least 15 consecutive nucleotides that differ from any one of the sequences shown in Table 2 by no more than 3 (0, 1, 2 or 3) nucleotides, and the sense strand has at least 15 nucleotides that are complementary to the antisense strand.

[0017] In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from any of the sequences shown in Table 2 by 0, 1, 2, or 3 nucleotides.

[0018] In some embodiments, the sense strand sequence is at least substantially complementary to the antisense strand sequence. In some preferred embodiments, the sense strand sequence is fully complementary to the antisense strand sequence (ie, 100% complementary).

[0019] In some embodiments, the length of the sense strand and the antisense strand are each independently 17-25 nucleotides; preferably, the length of the sense strand and the antisense strand are each independently 19-23 nucleotides; more preferably, the length of the sense strand and the antisense strand are each independently 19-21 nucleotides.

[0020] In some embodiments, the dsRNA comprises any one of the antisense or sense strand sequences in Table 2. In some embodiments, the dsRNA comprises any one of the sense or antisense strands shown in SEQ ID NOs: 223-478 or SEQ ID NOs: 513-520.

[0021] In some embodiments, the dsRNA comprises an antisense strand sequence and a sense strand sequence shown in the duplex sequence in Table 2.

[0022] In some embodiments, the antisense strand comprises completely contiguous nucleotides selected from SEQ ID NO: 224, 226, 228, 230, 232, 234, 236, 238, 256, 258, 260, 262, 272, 274, 276, 278, 448, 450, 452, 454, or 456.

[0023] In some embodiments, the sense strand comprises completely consecutive nucleotides selected from SEQ ID NO: 223, 225, 227, 229, 231, 233, 235, 237, 255, 257, 259, 261, 271, 273, 275, 277, 447, 449, 451, 453, or 455.

[0024] In some embodiments, the dsRNA comprises any duplex selected from C5-112 to C5-209 and C5-215 to C5-239.

[0025] In some embodiments, the dsRNA is selected from the following duplexes: C5-112, C5-113, C5-114, C5-115, C5-116, C5-117, C5-118, C5-119, C5-128, C5-129, C5-130, C5-131, C5-136, C5-137, C5-138, C5-139, C5-224, C5-225, C5-226, C5-227, and C5-228.

[0026] On the other hand, the present disclosure provides an RNAi agent (e.g., a dsRNAi agent) comprising any of the aforementioned dsRNAs and optionally comprising a targeting ligand. The targeting ligand is typically conjugated to the dsRNA and serves to target the RNAi agent to cells.

[0027] In some embodiments, the targeting ligand of the present disclosure is specifically targeted to the asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes. Preferably, the targeting ligand comprises N-acetyl-galactosamine (GalNAc), or the targeting ligand is a GalNAc derivative. More preferably, the targeting ligand is any targeting ligand disclosed in WO2022266753A1 (targeting moiety). Unless otherwise clearly contradictory, WO2022266753A1 is incorporated herein by reference in its entirety.

[0028] In some embodiments, the structure of the double-stranded RNAi agent is selected from Formula 1 or Formula 2, wherein R 2 is the dsRNA. According to common knowledge in the art, R 2 The dsRNA agent is conjugated to a targeting ligand via the 3' end or 5' end of the sense strand; preferably, the dsRNA agent is conjugated to the targeting ligand via the 3' end (eg, phosphate group) of the sense strand.

[0029] (abbreviated as: “R 2 -L01”)

[0030] (abbreviated as: “R 2 -L02”)

[0031] In some embodiments, the structure of the double-stranded RNAi agent is selected from any double-stranded RNAi agent (also known as dsRNA conjugate) in Table 3 or a pharmaceutically acceptable salt thereof.

[0032] In another aspect, the present disclosure also provides a cell, a vector, a host cell, and a pharmaceutical composition comprising the double-stranded RNAi agent of the present disclosure.

[0033] In some embodiments, the pharmaceutical composition comprises any of the aforementioned double-stranded RNAi agents or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be used to prevent and / or treat various corresponding diseases or conditions.

[0034] In some embodiments, the pharmaceutical composition is formulated for administration by injection or infusion, e.g., for intravenous, subcutaneous, intraperitoneal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0035] In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. A preferred buffered solution is phosphate-buffered saline (PBS).

[0036] In another aspect of the present disclosure, methods for inhibiting C5 expression in cells are also provided. These methods comprise contacting the cells with a double-stranded RNAi agent, dsRNA, or pharmaceutical composition disclosed herein, thereby degrading the mRNA transcript of the C5 gene, thereby inhibiting C5 gene expression in the cells. The inhibition can occur in vivo or in vitro.

[0037] In some embodiments, the cell is in a subject. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is an adipocyte. In some embodiments, the subject is a human.

[0038] In some embodiments, C5 expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0039] In another aspect, the present disclosure further provides a method for treating a subject having a condition mediated by C5 expression (e.g., a C5-related disease or condition), comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent or pharmaceutical composition of the present disclosure, thereby inhibiting C5 gene expression in cells.

[0040] In some embodiments, the subject is a human.

[0041] In some embodiments, the C5 expression-mediated disorder is selected from one or more of the following groups: paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), typical hemolytic uremic syndrome (tHUS), hemolytic uremic syndrome associated with Shiga toxin-producing Escherichia coli (E.Coli), myasthenia gravis (MG), rheumatoid arthritis (RA), Goodpasture's syndrome, glomerulonephritis (such as IgA nephropathy, membranous nephropathy, antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis), lupus nephritis, membranous proliferative glomerulonephritis (MPGN), etc.), Hashimoto's thyroiditis, ischemia-reperfusion injury, septic shock, neuromyelitis optica (NMO), antibody-mediated renal transplant rejection, Guillain-Barré syndrome (Guillain-Barré syndrome), etc. syndrome), Degos' syndrome, diabetic angiopathy, amyotrophic lateral sclerosis (ALS), dense deposit disease (DDD), Parkinson's disease (PD), autoimmune encephalitis, IgG4-related disease, asthma, antiphospholipid antibody syndrome, ischemia-reperfusion injury, multifocal motor neuropathy (MMN), multiple sclerosis (MS), thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, recurrent abortion, traumatic brain injury, cold agglutinin disease, dermatomyositis, graft dysfunction, myocardial infarction, sepsis, atherosclerosis, septic shock, spinal cord injury, psoriasis, macular degeneration, autoimmune hemolytic anemia (AIHA), antiphospholipid syndrome (APS), myocarditis, immune complex vasculitis, Takayasu's disease, and Kawasaki's disease (arteritis).

[0042] In some embodiments, the disorder mediated by C5 expression is paroxysmal nocturnal hemoglobinuria.

[0043] In some embodiments, the condition mediated by C5 expression is atypical hemolytic uremic syndrome.

[0044] In some embodiments, the condition mediated by C5 expression is IgA nephropathy. The IgA nephropathy includes primary IgA nephropathy and / or secondary IgA nephropathy. Secondary IgA nephropathy includes, but is not limited to, IgA nephropathy caused by systemic lupus erythematosus, Henoch-Schonlein purpura nephritis, hepatitis B virus-related nephritis, Crohn's disease, liver disease, tumors, and thrombotic thrombocytopenic purpura.

[0045] In some embodiments, the disorder mediated by C5 expression is rheumatoid arthritis.

[0046] In some embodiments, the condition mediated by C5 expression is myasthenia gravis.

[0047] In some embodiments, the expression of the C5 gene in the cell is inhibited so that the protein level of the C5 gene expression in the serum of the subject is reduced by at least 50%, 60%, 70%, 80%, 90% or 95% compared with that before the administration of the dsRNAi agent or pharmaceutical composition.

[0048] In some embodiments, the double-stranded RNAi agent or pharmaceutical composition is administered to a subject at a dose of about 0.10 mg / kg to about 50 mg / kg, for example, about 0.01 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0049] In some embodiments, the method further comprises determining the level of C5 in a sample from the subject. In some embodiments, the level of C5 in a sample from the subject is determined before, during, and / or after administration of the dsRNAi agent or pharmaceutical composition to the subject. Any suitable sample can be used, such as, but not limited to, a blood sample, a serum sample, or a liver tissue sample.

[0050] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent to treat the disorder mediated by C5 expression.

[0051] In some embodiments, the double-stranded RNAi agent of the present disclosure can be administered simultaneously or sequentially with another therapeutic agent. In some embodiments, the double-stranded RNAi agent is administered before or after administration of another therapeutic agent, such as a standard therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 shows the inhibition of C5 mRNA expression activity by unmodified RNAi duplexes at different concentration gradients.

[0053] Figure 2 shows the inhibition of C5 protein expression activity by unmodified RNAi duplexes at different concentrations; in the figure, for the 10nM, 0.1nM and 0.001nM groups, the results shown correspond to Mock, Non-coding, C5-Ref, C5-010, C5-013, C5-015, C5-016, C5-017, C5-038, C5-050, C5-051, C5-060, C5-068, and C5-069, respectively.

[0054] FIG3 shows the inhibition of C5 mRNA expression in HepG2 cells by unmodified and differently chemically modified RNAi duplexes at concentrations of 1 nM and 0.01 nM.

[0055] FIG4 shows the inhibition of C5 mRNA expression in Hep3B cells by unmodified and differently chemically modified RNAi duplexes at concentrations of 1 nM and 0.01 nM.

[0056] Figure 5 shows the inhibition of the expression activity of C5 protein levels secreted by Huh7 cells by unmodified and differently chemically modified RNAi duplexes at concentrations of 10 nM, 0.1 nM and 0.01 nM; in the figure, for the 10 nM, 0.1 nM and 0.01 nM groups, the results shown correspond to blank (Blank), NC, C5-Ref, C5-013, C5-116, C5-117, C5-118, C5-119, C5-015, C5-136, C5-137, C5-138, C5-139, C5-038, C5-128, C5-129, C5-130, C5-131, C5-051, C5-112, C5-113, C5-114, C5-115, and C5-Ref_CM, respectively.

[0057] Figure 6 shows the inhibition of the expression activity of C5 protein levels secreted by HepG2 cells by unmodified and differently chemically modified RNAi duplexes at concentrations of 10 nM, 0.1 nM and 0.01 nM; in the figure, for the 10 nM, 0.1 nM and 0.01 nM groups, the results shown correspond to blank (Blank), NC, C5-Ref, C5-013, C5-116, C5-117, C5-118, C5-119, C5-015, C5-136, C5-137, C5-138, C5-139, C5-038, C5-128, C5-129, C5-130, C5-131, C5-051, C5-112, C5-113, C5-114, C5-115, and C5-Ref_CM, respectively.

[0058] FIG7 and FIG8 show the inhibition of C5 mRNA expression activity by RNAi duplexes with different chemical modifications at different concentration gradients.

[0059] Figures 9, 11, 13 and 15 respectively show the expression levels of human C5 protein in the serum of humanized C5 mice after subcutaneous injection of different RNAi duplexes.

[0060] Figures 10, 12, 14 and 16 respectively show the expression levels of human C5 protein in the serum of humanized C5 mice after subcutaneous injection of different RNAi duplexes (normalized to the C5 expression level in the serum before injection as 100%).

[0061] FIG17 to FIG19 show the inhibition of C5 mRNA expression activity in Hep3B cells by RNAi duplexes with different chemical modifications at different concentration gradients.

[0062] FIG20 shows the changes in the levels of complement component C5 protein in the serum of cynomolgus monkeys at different time points after subcutaneous administration of 5 mg / kg or 25 mg / kg of C5-210.

[0063] FIG21 shows the percentage of complement classical pathway-mediated hemolysis remaining in cynomolgus monkey serum at various time points following subcutaneous administration of 5 mg / kg or 25 mg / kg of C5-210. DETAILED DESCRIPTION

[0064] The present disclosure provides RNAi agents and compositions that trigger RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of C5 target genes. The gene can be in cells, such as adipocytes and / or hepatocytes or other liver cells (or liver cells), such as cells in a subject (e.g., a human). The use of these RNAi agents and compositions enables targeted degradation of C5 mRNA in mammals. As inhibitors of C5 expression, the RNAi agents and compositions of the present disclosure can be used to prevent, treat and / or inhibit C5-related diseases or disorders, such as PNH and related disorders.

[0065] Thus, the present disclosure provides methods for treating, preventing, or inhibiting C5-related diseases or conditions, such as, but not limited to, PNH, aHUS, tHUS, MG, RA, glomerulonephritis, and the like, using RNAi compositions that trigger RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of C5 target genes.

[0066] The RNAi agents of the present disclosure comprise an antisense RNA strand having a region of up to about 30 nucleotides in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 19-22, 23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 15, 15-16, 15-17, 15-20, 15-21, 15-22, 15-23, 15-24 or at least 15 nucleotides, said region being substantially complementary to at least a portion of the transcript mRNA of the C5 target gene.

[0067] In certain embodiments, one or both strands of a double-stranded RNAi agent of the present disclosure are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides, and have a region of at least 15 consecutive nucleotides that is substantially complementary to at least a portion of the transcript mRNA of the C5 target gene. In some embodiments, these RNAi agents with longer antisense strands can, for example, include a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense and antisense strands form a double-stranded region (duplex) of 15-30 consecutive nucleotides.

[0068] The use of RNAi agents of the present disclosure enables targeted degradation of C5 mRNA in mammals. The inventors have confirmed that the RNAi agents of the present disclosure can trigger the cleavage of C5 RNA transcripts mediated by RNA-induced silencing complex (RISC), thereby resulting in significant inhibition of the expression of C5 target genes. In certain embodiments, the RNAi agents of the present disclosure are more effective (e.g., more potent) and / or more specific (e.g., safer, with fewer off-target effects) than previous RNAi agents targeting the same gene. In certain embodiments, RNAi agents are targeted to specific sites in C5 mRNA by selecting them, and / or RNA modifications are included to increase efficacy, effectiveness, specificity, and / or safety. Methods and compositions comprising these RNAi agents can be used to treat subjects suffering from C5-related diseases or conditions. Therefore, the present disclosure provides methods for treating, preventing, or suppressing C5-related diseases or conditions in subjects who will benefit from using RNAi agents and compositions of the present disclosure to suppress or reduce C5 expression.

[0069] The present disclosure also provides methods for preventing at least one symptom in a subject having a condition that would benefit from inhibition or reduction of C5 expression. The following detailed description will disclose how to prepare and use RNAi agents and compositions thereof to inhibit the expression of C5 target genes, as well as compositions, uses, and methods for treating subjects who would benefit from inhibition and / or reduction of C5 target gene expression, such as subjects susceptible to or diagnosed with PNH, aHUS, tHUS, MG, RA, or glomerulonephritis.

[0070] definition

[0071] In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0072] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Similarly, the word "another" can mean at least a second or more.

[0073] The term "or" is used herein to mean the term "and / or" and can be used interchangeably with the term "and / or" unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand, or sense strand and antisense strand."

[0074] As used in this specification and claims, the words "comprising" (and any forms of including, such as "comprises" and "including"), "having" (and any forms of having, such as "have" and "having"), "including" (and any forms of including, such as "including" and "including"), and "comprising" (and any forms of including, such as "containing" and "including"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0075] The term "about" or "approximately" is used to indicate that the value includes the error introduced by the instruments and methods used in determining the value. The term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods. When "about" precedes a series of numbers or a range, it should be understood that "about" can modify each number in the series or range.

[0076] The terms "at least," "not less than," or "or more" preceding a number or a range of numbers should be understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that can logically be included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 15 nucleotides in a 17-nucleotide nucleic acid molecule" means 15, 16, or 17 nucleotides having the specified properties. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in the series or range.

[0077] As used herein, "not more than" or "or less" is understood to refer to the value adjacent to the phrase and logically lower values ​​or integers, as is logical from the context, to zero. For example, a duplex having an overhang of "not more than 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "not more than" appears before a series of numbers or a range, it is understood that "not more than" can modify each number in the series or range. As used herein, a range includes an upper limit and a lower limit.

[0078] The term "C5" means "complement component C5", which is a well-known gene, polypeptide and / or protein, also known in the art as "complement C5", C5D, C5a, C5b, CPAMD4, ECLZB, "complement component 5" or "complement C5". In the present disclosure, C5 mRNA refers to the mRNA having the sequence shown in GenBank Accession No. NM_001735.3. The term "target sequence" or "target nucleic acid" or "target mRNA" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during the transcription of the target gene. In one embodiment, the target sequence is located in the protein coding region of the target gene. In another embodiment, the target sequence is located in the 3'UTR of the target gene. The target nucleic acid can be a cellular gene (or an mRNA transcribed from the gene) whose expression is associated with a particular condition or disease state. In certain embodiments, the target sequence is 15-30 nucleotides in length, for example, 15-23 nucleotides in length, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 17-30, 17-29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-23, 17-22, 17-21, 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16-22, or 16-21 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also considered to be part of this disclosure.

[0079] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0080] The terms "siRNA," "RNAi agent," "siRNA agent," and "RNA interference agent" are used interchangeably herein and refer to biologically active agents that contain RNA and mediate targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. RNAi agents direct the specific degradation of mRNA sequences through a process known as RNA interference. RNAi agents modulate (e.g., inhibit) the expression of genes in cells, such as cells in a subject (e.g., a mammalian subject, such as a human). In some embodiments, the RNAi agent used in the compositions, uses, and methods of the present disclosure comprises a double-stranded RNA (dsRNA) or duplex of the present disclosure and may be referred to herein as a "double-stranded RNAi agent," "dsRNAi agent," or "dsRNA agent."

[0081] In certain embodiments, the dsRNAi agent of the present invention includes a double-stranded RNA agent, which, when introduced into a cell, is processed into short interfering RNA by a nuclease called Dicer. Short interfering RNA is integrated into RISC, and one or more helicases unwind the RNA duplex, allowing the complementary antisense strand to guide target recognition. After binding to the target mRNA, one or more nucleases in RISC will cut the target mRNA to induce silencing. Therefore, in other embodiments, the siRNA agent relates to single-stranded RNA produced in the cell and promotes the formation of the RISC complex to achieve the silencing of the target gene. In some such embodiments, the RNAi agent is a single-stranded siRNA (ssRNAi), which can be introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC nuclease, Argonaute2, and then cuts the target mRNA. The ssRNAi agent is generally 15-30 nucleotides in length and can be chemically modified. Any antisense oligonucleotide described herein can be used as the ssRNAi agent described herein.

[0082] The term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid chains, with "sense" (or "justice") and "antisense" orientations relative to the target RNA. In some embodiments of the present disclosure, dsRNA triggers the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. Generally speaking, the majority of the nucleotides in each chain of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both chains may also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Each chain of a dsRNA molecule may have a length ranging from 12 to 40 nucleotides. For example, each strand can be between 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 17-25 nucleotides in length, 17-22 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, or 21-23 nucleotides in length, and the sense and antisense strands can be equal or unequal lengths without limitation.

[0083] The term "antisense strand" refers to a strand of an iRNA (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence (e.g., C5 mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence. Where the complementary region is not completely complementary to the target sequence, there may be mismatches in the interior or terminal regions of the molecule. Typically, the most tolerated mismatches are present in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5'- and / or 3'-ends of the dsRNA. The antisense and sense strands of a dsRNA may have the same or different lengths, as are known in the art.

[0084] When a first sequence is referred to as being "substantially complementary" to a second sequence, the two sequences can be fully complementary (i.e., complementary over the entire length of one or both nucleotide sequences), or they can form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization over a distance of up to 30 base pairs, while retaining the ability to hybridize under appropriate conditions (conditions relevant to their application, such as inhibition of gene expression, such as physiological conditions). It should be noted that when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, can be referred to as "fully complementary" for the purposes described herein.

[0085] The term "sense strand" refers to the strand of a dsRNA that comprises a region that is substantially complementary to a region of the antisense strand.

[0086] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Such conditions can, for example, be stringent conditions, wherein stringent conditions can include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may also be applicable. For example, complementary sequences are sufficient to enable the relevant function of the nucleic acid, such as RNAi. A skilled person can determine the set of conditions that is most suitable for testing the complementarity of two sequences based on the ultimate use of the hybridizing nucleotides.

[0087] The terms "complementary," "fully complementary," and "substantially complementary" as used herein may be used to describe base matching between the sense and antisense strands of a dsRNA, or between two oligonucleotides or polynucleotides, such as the antisense strand of a dsRNA agent and a target sequence, and their meanings will be understood from the context in which the terms are used.

[0088] In the present disclosure, "g," "c," "a," and "u" mean non-modified conventional nucleotides, which represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively.

[0089] The term "modified nucleotide" refers to any nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term "modified nucleotide" encompasses substitution, addition, or removal of, for example, a functional group or atom of an internucleoside linkage, a sugar moiety, or a nucleobase. In the present disclosure, capitalization alone indicates 2'-fluoro modifications (i.e., A, C, G, U), capitalization plus m indicates 2'-O-methyl-modifications (i.e., Am, Cm, Gm, Um), capitalization plus d indicates 2'-deoxy modifications (i.e., Ad, Cd, Gd, Ud, Td), s indicates 3'-phosphorothioate modifications, and VP indicates 5'-vinyl phosphate modifications. Non-limiting examples of common modified nucleotides and related moieties are defined as shown in Table A.

[0090] Table A. Definitions of Exemplary Modified Nucleotides

[0091] The term "derivative" as used in the present disclosure should be understood as another compound that is similar in structure but different in some minor structures.

[0092] The term "inhibit" and similar expressions refer to reducing or effectively stopping, and can be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and include any level of inhibition. As a non-limiting example, "inhibit" herein refers to reducing or effectively reducing the onset or progression of a C5-related disease in a subject, including a reduction in one or more aspects of the disease (e.g., symptoms, tissue characteristics, cellular activity, inflammatory activity, or immune activity, etc.), or the absence of detectable worsening.

[0093] The term "C5-associated disease or condition" or "condition mediated by C5 expression" includes any disease or condition caused, mediated, or associated with C5 gene expression or protein production, and includes any disease or condition that would benefit from or be ameliorated by a reduction in C5 gene expression or protein activity.

[0094] As used herein, "inhibited expression" of a gene (e.g., C5) refers to a decrease in the amount or level of RNA transcript (e.g., C5 mRNA) or protein encoded by the gene and / or a decrease in the amount or level of activity of the gene in a cell, cell population, sample, or subject, as compared to an appropriate reference (e.g., a reference cell, cell population, sample, or subject). As used herein, "inhibiting C5 expression" refers to a decrease in the amount or level of C5 mRNA and / or C5 protein (or polypeptide) in a cell, cell population, sample or subject, such as an inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, compared to an appropriate reference (e.g., a reference cell, cell population, sample or subject).

[0095] As used herein, the phrase "contacting cells with an RNAi agent" (e.g., a dsRNAi agent) includes contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or contacting cells with an RNAi agent in vivo. The contact can be performed directly or indirectly. Therefore, for example, the RNAi agent can be physically contacted with the cell, or alternatively, the RNAi agent can be placed in a situation where it will allow or cause it to subsequently contact the cell. Contacting cells in vitro can be performed by, for example, incubating the cell with the RNAi agent. Contacting cells in vivo can be performed, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area (e.g., bloodstream or subcutaneous space) so that the RNAi agent subsequently reaches the tissue where the cell is to be contacted. For example, the RNAi agent can contain or be coupled to a targeting ligand, such as GalNAc, which guides the RNAi agent to a site of interest, such as the liver. A combination of in vitro and in vivo contact methods is also possible. For example, cells can also be contacted with an RNAi agent of the present invention in vitro and subsequently transplanted into a subject. In certain embodiments, contacting cells with RNAi agents includes promoting or influencing the uptake or absorption of cells. The absorption or uptake of RNAi agents can occur by unassisted diffusion or active cellular processes, or by adjuvants or devices. The introduction of RNAi agents into cells can be performed in vitro or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection.

[0096] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. In certain embodiments, the subject is a human.

[0097] In some embodiments, "treatment" of any disease or condition refers to improving at least one disease or condition. In certain embodiments, "treatment" refers to improving at least one physical parameter, which may or may not be perceived by the patient. In certain embodiments, "treatment" refers to inhibiting a disease or condition physically (e.g., stabilization of overt symptoms), physiologically (e.g., stabilization of physical parameters), or both. In some embodiments, "treatment" refers to improving the quality of life of a subject in need or reducing the symptoms or side effects of a disease. A "therapeutically effective amount" refers to the amount of a dsRNA or dsRNAi agent that is sufficient to achieve the treatment or prevention of a disease when administered to a cell, tissue, or subject alone or in combination with other therapeutic agents. A "therapeutically effective amount" will vary depending on the compound or RNAi agent, the disease and its severity, and the age, weight, etc. of the subject suffering from the disease to be treated or prevented. As used herein, the term "therapeutically effective amount" refers to an amount of a compound or composition sufficient to prevent, treat, inhibit, reduce, ameliorate, or eliminate one or more causes, symptoms, or complications of a disease or condition, such as PNH, aHUS, tHUS, MG, RA, or glomerulonephritis. The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein. When an active ingredient is administered alone to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients, whether administered in combination, sequentially, or simultaneously, that results in a therapeutic effect. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0098] In some embodiments, "prevention" or "prevention" of any disease or condition refers to at least reducing the likelihood of the risk (or susceptibility) of acquiring the disease or condition (i.e., so that at least one clinical symptom of the disease does not appear in a patient who may be exposed to or susceptible to the disease but has not yet experienced or displayed symptoms of the disease). When used with respect to a disease, disorder, or condition that would benefit from a decrease in C5 expression, it means reducing the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as PNH, aHUS, tHUS, MG, RA, or glomerulonephritis. Failure to develop a disease, disorder, or condition, or a reduction in the development of symptoms associated with the disease, disorder, or condition (e.g., a reduction of at least about 10% for the disease or condition on a clinically accepted scale), or a delay in the manifestation of symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0099] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0100] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material (involved in carrying or transporting a compound or dsRNAi agent from one organ or part of the body to another organ or part of the body).

[0101] dsRNA and RNAi agents

[0102] In one aspect, dsRNA of the present disclosure comprises at least two nucleotide sequences, sense sequence and antisense sequence.In some embodiments, sense strand is selected from the sequence provided in any one of Table 2, and the corresponding antisense strand of sense strand is selected from the sequence provided in any one of Table 2.In this respect, one of the two sequences is complementary to another of the two sequences, wherein one of the sequences is substantially complementary to the mRNA sequence produced in the expression of the relevant target gene.Therefore, in this respect, dsRNA will comprise two oligonucleotides, wherein one of the oligonucleotides is described as any one of the sense strands in Table 2, and second oligonucleotides is described as the corresponding antisense strand of any one of the sense strands in Table 2.Should be understood that dsRNA of the present disclosure is fully modified substantially.

[0103] In some embodiments, the dsRNA comprises any one of the sense strands or antisense strands set forth in SEQ ID NOs: 223-478 or SEQ ID NOs: 513-520.

[0104] In some embodiments, the antisense strand comprises completely contiguous nucleotides selected from SEQ ID NO: 224, 226, 228, 230, 232, 234, 236, 238, 256, 258, 260, 262, 272, 274, 276, 278, 448, 450, 452, 454, or 456.

[0105] In some embodiments, the sense strand comprises completely consecutive nucleotides selected from SEQ ID NO: 223, 225, 227, 229, 231, 233, 235, 237, 255, 257, 259, 261, 271, 273, 275, 277, 447, 449, 451, 453, or 455.

[0106] In some embodiments, the dsRNA comprises an antisense strand sequence and a sense strand sequence shown in the duplex sequence in Table 2.

[0107] In some embodiments, the dsRNA comprises any duplex selected from C5-112 to C5-209 and C5-215 to C5-239.

[0108] In some embodiments, the dsRNA is selected from the following duplexes: C5-112, C5-113, C5-114, C5-115, C5-116, C5-117, C5-118, C5-119, C5-128, C5-129, C5-130, C5-131, C5-136, C5-137, C5-138, C5-139, C5-224, C5-225, C5-226, C5-227, and C5-228.

[0109] It is well known to those skilled in the art that dsRNAs having a duplex structure of approximately 20 to 23 base pairs (e.g., 21 base pairs) are considered particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). In some embodiments, the dsRNA can include at least one strand that is at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes of the dsRNAs listed in Table 2, with only a few nucleotides removed from one or both ends, compared to the above-described dsRNAs, can also have similar effects. Thus, dsRNAs having a sequence of at least 12, 13, 14, 15, 19, 20 or more consecutive nucleotides derived from any one of Table 2, and whose ability to inhibit C5 gene expression does not differ by more than about 5%, 10%, 15%, 20%, 25% or 30% from a dsRNA comprising the entire sequence, are considered to be within the scope of the present disclosure.

[0110] The dsRNA of the present disclosure can be synthesized by standard methods known in the art. Double-stranded RNAi compounds of the present disclosure can be prepared using a two-step procedure. First, each chain of the double-stranded RNA molecule is prepared separately and then annealed. Each chain of the siRNA compound can be prepared using solution phase or solid phase organic synthesis or both. The advantage of organic synthesis is that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

[0111] On the other hand, the present disclosure provides an RNAi agent (e.g., a dsRNAi agent) comprising any of the aforementioned dsRNAs and optionally comprising a targeting ligand. The targeting ligand is typically conjugated to the dsRNA and serves to target the RNAi agent to cells.

[0112] In certain embodiments, the dsRNAi agent of the present disclosure is further modified by covalently linking one or more conjugate groups. Generally speaking, the conjugate group changes one or more properties of the dsRNA agent of the present disclosure connected, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cellular uptake, charge and removal. Conjugate groups are commonly used in the field of chemistry, and are directly or by optional linking moieties or linking groups connected to the parent compound. Conjugate groups preferably include but not limited to polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dye.

[0113] In some embodiments, the targeting ligand of the present disclosure comprises N-acetyl-galactosamine (GalNAc), or a GalNAc derivative, such as L96 (see siRNA drug Inclisiran). In some embodiments, the targeting ligand is any targeting ligand disclosed in WO2022266753A1. Unless otherwise clearly contradictory, WO2022266753A1 is incorporated herein by reference in its entirety.

[0114] In some embodiments, the structure of the dsRNAi agent is selected from Formula 1 or Formula 2, wherein R 2 According to common knowledge in this field, R 2 The dsRNAi agent is formed by conjugating the 3' end or 5' end of the sense strand of the dsRNA to a targeting ligand.

[0115] In some embodiments, the dsRNAi agent is any double-stranded RNAi agent (also known as a dsRNA conjugate) selected from Table 3, or a pharmaceutically acceptable salt thereof.

[0116] The present disclosure also encompasses various salts, mixed salts, and free acid forms of the dsRNAi agents. In some embodiments, the dsRNAi agent is in free acid form. In some embodiments, the dsRNAi agent is in salt form. In one embodiment, the dsRNAi agent is in sodium salt form. According to common knowledge in the art, when the dsRNAi agent of the present disclosure is in sodium salt form, sodium ions are present in the agent as counterions to the phosphodiester and / or phosphorothioate groups.

[0117] Delivery and Use of RNAi Agents

[0118] The RNAi agents of the present invention can be delivered to cells, such as cells in a subject (e.g., a subject with a metabolic disorder) in a variety of ways. For example, delivery can be carried out by contacting cells with the RNAi agents of the present invention in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition (e.g., a pharmaceutical composition) comprising an RNAi agent (e.g., dsRNA) to the subject. Alternatively, in vivo delivery can be carried out indirectly by administering one or more vectors that encode and guide the expression of the RNAi agent.

[0119] In one embodiment, the cells are liver cells, such as hepatocytes. In one embodiment, the cells are adipocytes. In certain embodiments, the RNAi agent is taken up by one or more tissue or cell types present in an organ (e.g., liver, adipose tissue).

[0120] Another aspect of the present disclosure relates to a method for reducing the expression and / or activity of a C5 gene in a subject, comprising administering a dsRNAi agent of the present disclosure to the subject. In some embodiments, the method comprises administering a therapeutically effective amount of a dsRNAi agent of the present disclosure to the subject, thereby inhibiting or reducing the expression of the C5 gene in the subject (e.g., a cell in the subject). In some embodiments, the method comprises contacting a cell with a double-stranded RNAi agent of the present disclosure such that the expression of the C5 gene is inhibited or reduced in the cell. In some such embodiments, the mRNA transcript of the target gene, such as the C5 gene, is degraded in the subject or cell, thereby inhibiting or reducing the expression of the C5 gene in the subject or cell.

[0121] In another aspect, the disclosure relates to methods of treating a subject having or at risk of having a C5-related disorder or at risk of developing a metabolic disorder, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent of the disclosure, thereby treating the subject.

[0122] In another aspect, the present disclosure relates to a method of treating or preventing a C5-associated disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent such that the C5-associated disease or disorder is treated or prevented. Examples of C5-associated diseases or conditions include, but are not limited to, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), typical hemolytic uremic syndrome (tHUS), hemolytic uremic syndrome associated with Shiga toxin-producing Escherichia coli (E. Coli), myasthenia gravis (MG), rheumatoid arthritis (RA), Goodpasture's syndrome, glomerulonephritis (e.g., IgA nephropathy, membranous nephropathy, antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis), lupus nephritis, membranoproliferative glomerulonephritis (MPGN), etc.), Hashimoto's thyroiditis, ischemia-reperfusion injury, septic shock, neuromyelitis optica (NMO), antibody-mediated renal transplant rejection, Guillain-Barré syndrome (GBS), and nephrotic syndrome. syndrome), Degos' syndrome, diabetic vasculopathy, amyotrophic lateral sclerosis (ALS), dense deposit disease (DDD), Parkinson's disease (PD), autoimmune encephalitis, IgG4-related disease, asthma, antiphospholipid antibody syndrome, ischemia-reperfusion injury, multifocal motor neuropathy (MMN), multiple sclerosis (MS), thrombotic thrombocytopenic purpura (TTP), spontaneous abortion, recurrent abortion, traumatic brain injury, cold agglutinin disease, dermatomyositis, graft dysfunction, myocardial infarction, sepsis, atherosclerosis, septic shock, spinal cord injury, psoriasis, macular degeneration, autoimmune hemolytic anemia (AIHA), antiphospholipid syndrome (APS), myocarditis, immune complex vasculitis, Takayasu's disease, and Kawasaki's disease (arteritis).

[0123] In some embodiments, the subject is a human.

[0124] In some embodiments, the subject suffers from PNH, aHUS, tHUS, MG, RA, IgA nephropathy, or glomerulonephritis. The IgA nephropathy includes primary IgA nephropathy and / or secondary IgA nephropathy. The secondary IgA nephropathy includes, but is not limited to, systemic lupus erythematosus, Henoch-Schonlein purpura nephritis, hepatitis B virus-related nephritis, Crohn's disease, liver disease, tumors, and IgA nephropathy caused by thrombotic thrombocytopenic purpura. The pathological features of the IgA nephropathy include one or more of mesangial cell hyperplasia, capillary endocellular hyperplasia, segmental glomerulosclerosis and / or adhesions, tubular atrophy, interstitial fibrosis, and crescent formation.

[0125] In some embodiments, the subject is a human.

[0126] In some embodiments of the present disclosure, expression of the C5 gene in a subject or cell reduces C5 protein levels in the subject's serum by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0127] In some embodiments, inhibiting the expression of the C5 gene in a cell reduces the protein level of the C5 gene expression in the serum of the subject by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0128] In some embodiments of the present disclosure, the dsRNAi agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg, or at a dose of about 0.10 mg / kg to about 50 mg / kg, for example, but not limited to, a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 50 mg / kg, about 5 mg / kg to about 50 mg / kg, about 10 mg / kg to about 30 mg / kg, about 10 mg / kg to about 20 mg / kg, about 15 mg / kg to about 20 mg / kg, about 15 mg / kg to about 25 mg / kg, about 15 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 30 mg / kg.

[0129] In some embodiments of the present disclosure, the method further comprises determining the level of C5 in a sample from the subject, e.g., in a blood, serum, liver tissue, or adipose tissue sample. The level of C5 in a sample from the subject can be determined before, during, and / or after administration of a dsRNAi agent to the subject (e.g., to monitor efficacy or treatment efficiency, monitor C5 mRNA and / or protein levels before, during, or after treatment, etc.).

[0130] In some embodiments of the present disclosure, the method further comprises administering to the subject an additional therapeutic agent to treat a disease or condition associated with C5. The therapeutic agent includes, but is not limited to, eculizumab, ravulizumab, crovalimab, narsoplimab, prednisolone, hydrocortisone, warfarin, heparin, aspirin, dipyridamole, dexamethasone, cyclophosphamide, neostigmine, celecoxib, methotrexate, tacrolimus, azathioprine, mycophenolate mofetil, cyclosporine, hydrochlorothiazide, budesonide, atrasentan, ambrisentan, zilucoplan, avacopan, iptacopan, danicopan, rituximab, belimumab, and a combination of any of the foregoing drugs.

[0131] In some embodiments, the dsRNAi agent of the present disclosure is administered by injection or by infusion. In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In one embodiment, the double-stranded RNAi agent is administered intramuscularly. In one embodiment, the double-stranded RNAi agent is administered intravenously. In one embodiment, the double-stranded RNAi agent is administered by systemic administration to the lungs, such as intranasal administration or oral inhalation administration.

[0132] In some embodiments, the pharmaceutical composition comprises a dsRNAi agent of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be used in practice for the prevention and / or treatment of various corresponding diseases or conditions. An acceptable carrier (or excipient) is a substance that is intentionally included in a drug delivery system in addition to an active pharmaceutical ingredient (API, therapeutic product, such as a dsRNA agent of the present invention). The carrier or excipient does not or is not intended to exert a therapeutic effect at the intended dose. The carrier or excipient may play the following roles: a) aids in the handling of the drug delivery system during preparation, b) protects, supports or enhances the stability, bioavailability or patient acceptability of the API; c) aids in product identification; and / or d) enhances any other properties of the overall safety, efficacy or delivery of the API during storage or use.

[0133] Carriers or excipients include, but are not limited to, the following components: absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavorings, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, and wetting agents.

[0134] In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. In some embodiments, the buffered solution is phosphate buffered saline (PBS).

[0135] The present disclosure includes all combinations of the specific embodiments described. Further embodiments of the present disclosure and the full scope of applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present disclosure, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0136] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the specific embodiments with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples disclosed herein.

[0137] Example

[0138] Example 1: dsRNA synthesis

[0139] 1.1 Target sequence screening

[0140] dsRNAs were designed based on the complete C5 mRNA sequence, all of which were obtained from the NCBI gene database (https: / / www.ncbi.nlm.nih.gov / gene / ). All dsRNAs were designed to ensure full identity to human and cynomolgus macaque sequences (human: NM_001735.3 (SEQ ID NO: 525); cynomolgus macaque: XM_005580915.3 (SEQ ID NO: 526)) in a comprehensive search of the appropriate transcriptome (defined as the set of NM_ and XM_ records within the human or cynomolgus macaque NCBI Refseq groups).

[0141] The entire sequence was scanned to obtain all potential dsRNA sequences 19-23 nucleotides long and simultaneously compared to the cynomolgus macaque sequence to ensure a match. All human / cynomolgus macaque sequences were then BLAST-aligned against the human full transcriptome mRNA sequence, and any dsRNAs with potential off-target effects were removed. The activity of all dsRNAs was evaluated using rational dsRNA design principles, and molecules with low theoretical activity were removed.

[0142] 1.2dsRNA synthesis

[0143] dsRNAs were designed for different regions of human C5 (dsRNA sequences are shown in Tables 1 and 2 ) and synthesized and annealed by Suzhou Beixin Biotechnology Co., Ltd.

[0144] Table 1: Sense and antisense strand sequences of unmodified C5-dsRNA reagents

[0145] Table 2: Sense and antisense strand sequences of modified C5-dsRNA agents

[0146] 1.3 RNAi synthesis

[0147] Using the GalNAc-coupled immobilized carrier (CPG or PS) in Formulas 1 and 2, fully modified siRNAs were designed according to the original sequences and synthesized and annealed by Suzhou Beixin Biotechnology Co., Ltd. The sense and antisense strand sequences of these dsRNAi agents are shown in Table 3.

[0148] The synthesis of the targeting ligands in Formula 1 and Formula 2 refers to WO2022266753A1.

[0149] Table 3: Sense and antisense strand sequences of modified C5-dsRNAi agents

[0150] In addition, control dsRNAi agents were prepared with reference to Examples 1.2 and 1.3, and their specific sequences are shown in Table 4. Wherein, L96 is a GalNAc ligand well known in the art.

[0151] Table 4: Sense and antisense strand sequences of control dsRNAi agents

[0152] Example 2: In vitro activity detection

[0153] 2.1 Fluorescence quantitative PCR

[0154] 2.1.1 Cell culture and transfection

[0155] Hep3B cells were cultured in EMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and double antibodies (Gibco), and HepG2 cells were cultured in EMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and double antibodies (Gibco) at 5% CO2 and 37°C. After the cells grew to almost completely cover the culture flask, they were digested with trypsin and resuspended. The resuspended cells were adjusted in density and seeded into 96-well plates at 3e4 / well, and dsRNA transfection complexes were added to the suspension. The dsRNA transfection complex was obtained by mixing Opti-MEM (Gibco) containing 0.3 μL / well liposome RNAi MAX (Thermo) with a 1:1 mixture of the dsRNA mixture. After 48 hours of cell culture, Dynabeads was used according to the instructions. TM Total mRNA was extracted using an mRNA isolation kit (Thermo). mRNA was eluted with 20 μL of RNase-free H₂O at 80°C for 5 minutes, and 15 μL of the supernatant was quickly transferred to a magnetic rack. Heating was performed using a BIO-RAD T100 Thermal Cycler PCR instrument.

[0156] 2.1.2 cDNA reverse transcription

[0157] cDNA synthesis was performed using the Vazyme Reverse Transcription Kit. 2 μL of the 15 μL supernatant removed from Example 2.1.1 was added to 1 μL of Oligo(dT)20VN (poly-dT), 2 μL of reverse transcriptase, and 2 μL of 10× RT Mix. The volume was then brought to 20 μL using RNase-free HO. The reaction was then completed using a BIO-RAD T100 Thermal Cycler at 37°C for 15 minutes and 85°C for 5 seconds. After completion, the reaction was stored at 4°C.

[0158] 2.1.3 Fluorescence quantitative PCR

[0159] The relative mRNA levels of C5 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were detected using the SYBR green method (TIANGEN). The instrument used was the LightCycler 480II (Roche) fluorescence quantitative system. The reaction conditions were: (1) 50°C, 15 minutes; (2) pre-denaturation at 95°C, 15 minutes; (3) denaturation at 95°C, 10 seconds, annealing and extension at 60°C, 30 seconds. Step (3) lasted for 40 cycles. The results were normalized with the negative control to obtain the relative mRNA levels and knockdown efficiency. If IC50 was required, it was obtained by four-parameter fitting using Graphpad Prism. The results are shown in Figures 1, 3, 4, 7, 8 and 17-19. The data in the same figure are the test results of the same batch.

[0160] Example 3: ELISA detection of the content of C5 secreted by cells

[0161] 3.1 ELISA detection of C5 content

[0162] Huh7 cells (ATCC) were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and a double-antibody (Gibco) in an atmosphere of 5% CO₂ at 37°C. Once the cells had grown to nearly cover the flask, they were trypsinized and resuspended. The resuspended cells were seeded at a density of 2e4 cells / well in a 96-well plate, and the dsRNA transfection complex was added to the suspension. For the single-dose screening of unmodified dsRNA, 10nM, 0.1nM and 0.001nM dsRNA were selected for transfection. For the multi-dose screening, 10.00000nM, 3.33333nM, 1.11111nM, 0.3707nM, 0.12346nM, 0.04115nM, 0.01372nM, 0.00457nM, 0.00152nM and 0.00051nM dsRNA were selected for transfection. For the single-dose screening of modified dsRNA, 10nM, 0.1nM and 0.01nM dsRNA were selected. dsRNA was transfected, and multiple dose screening selected 10.00000nM, 3.33333nM, 1.11111nM, 0.370.7nM, 0.12346nM, 0.04115nM, 0.01372nM, 0.00457nM, 0.00152nM, and 0.00051nM dsRNA for transfection. The dsRNA transfection complex consisted of a 1:1 mixture of Opti-MEM (Gibco) containing 0.3μL / well lipofectamine RNAiMAX (Thermo) and the dsRNA mixture. After 24 hours of cell culture, the medium was replaced with Opti-MEM (Gibco). After an additional 72 hours of culture, the culture supernatant was collected and assayed for C5 protein content using a commercial ELISA kit (R&D systems, DY2037). All cell supernatant samples were thoroughly thawed, diluted 4-fold in Opti-MEM, and added to an ELISA plate coated with the capture antibody. After incubation at room temperature for 2 hours, the plate was washed and HRP-labeled detection antibody was added and incubated at room temperature for 1 hour. After washing, the plate was developed with TMB (Thermo) and absorbance at 450 nm was measured using a multi-function microplate reader. A standard curve was fitted using four parameters and used to convert the C5 protein concentrations of the samples. The results are shown in Figures 2, 5, and 6.

[0163] Example 4: Stem-loop PCR

[0164] 4.1 Stem-loop PCR

[0165] The stem-loop method has been widely used to determine the absolute concentrations of dsRNA and miRNA (Curr Protoc Mol Biol. 2011 Jul; Chapter 15: Unit 15.10). Stem-loop primers are designed for different dsRNAs and used instead of oligo dT for reverse transcription. dsRNA concentrations are then calculated using a standard curve using quantitative PCR. Reverse transcription (TIANGEN): (1) Sample pretreatment: 1 μL sample, 1 μL Stem-loop primer, 1 μL dNTP, then filled up to 10 μL with RNase-free H2O. The conditions were: 1) 85°C, 5 minutes; 2) 60°C, 5 minutes. (2) Reverse transcription: 10 μL pretreated sample, 4 μL 5X buffer, 1 μL RT Enzyme Mix, 0.5 μL R Nasin, then filled up to 10 μL with RNase-free H2O. The conditions were: 1) 16°C, 30 minutes; 2) 42°C, 60 minutes; 3) 95°C, 5 minutes. Fluorescence quantitative PCR (TIANGEN) reaction conditions were: (1) 95°C, 15 minutes; (2) 95°C, 5 seconds, 60°C, 10 seconds, 72°C, 1 second. Step (2) was repeated for 40 cycles.

[0166] 4.2 dsRNA stability testing

[0167] 4.2.1 FBS stability

[0168] (1) Dilute dsRNA to 2 μM, pipette 4 μL into 76 μL FBS stock solution, and mark it as zero point. (The final concentration of dsRNA is 100 nM, and the matrix ratio is ≥90%); (2) Pipette 10 μL from zero point into a new tube, mark the corresponding time point, and incubate at 37°C. (8-tube strips can be used for sample preparation; the time points are 0, 6 hours, and 24 hours, and the incubation at 37°C can be placed in a CO2 incubator); (3) Remove the corresponding sample according to the incubation time, quickly freeze it in liquid nitrogen prepared in advance, and then store the sample at -20°C. The sample will be tested using the 3.1 Stem-loop PCR method.

[0169] 4.2.2 Human Liver S9 Stability

[0170] (1) Dilute dsRNA to 2 μM, draw 4 μL and add it to 76 μL of human liver S9 stock solution (20 mg / mL), marking it as zero point. (The final concentration of dsRNA is 100 nM, and the matrix ratio is ≥90%); (2) Draw 10 μL from zero point and add it to a new tube, mark the corresponding time point, and incubate at 37°C. (8 strips can be used for sample preparation; the time points are 0, 6h, 24h, and incubation at 37°C can be placed in a CO2 incubator); (3) Take out the corresponding sample according to the incubation time and quickly freeze it in liquid nitrogen prepared in advance. The sample is then stored at -20°C and subsequently detected using the 3.1 Stem-loop PCR method.

[0171] 4.2.3 Stability of human liver homogenate

[0172] (1) Prepare 500 mg / mL liver homogenate with PBS at 4°C and store at -80°C for subsequent sample incubation; (2) Dilute dsRNA to 2 μM, dilute dsRNA to 100 nM with liver homogenate and mark it as zero point, with the matrix ratio ≥ 90%; (3) Pipette 10 μL from zero point into a new sample tube, mark the corresponding time point, and incubate at 37°C for different time periods. (4) Remove the corresponding sample according to the incubation time and quickly freeze it in liquid nitrogen. The sample was then stored at -80°C and centrifuged at high speed before use to remove the matrix effect. The zero point group sample gradient dilution was used as the standard curve and detected using the 3.1 Stem-loop PCR method.

[0173] Example 5: Free intake

[0174] In this system, different concentrations of dsRNA were co-incubated with primary cynomolgus monkey hepatocytes for a certain period of time, and then the cell mRNA was extracted. The mRNA level was detected by real-time fluorescence quantitative PCR to evaluate the biological activity of dsRNA taken up by primary cynomolgus monkey hepatocytes and the specific degradation of mRNA. Primary cynomolgus monkey hepatocytes were obtained from Miaotong (Shanghai) Biotechnology Co., Ltd. and revived using the matching recovery medium. After washing, they were resuspended in the maintenance medium and adjusted to a density of 3e4 / well and inoculated into a 96-well plate coated with the matching coating medium. After the cells were completely attached, dsRNA was added, and after the cells were cultured for a certain period of time, Dynabeads were used according to the instructions. TM Total mRNA was extracted using an mRNA isolation kit (Thermo). mRNA was eluted with 20 μL of ddH₂O at 80°C for 5 minutes, and 15 μL of the supernatant was quickly transferred to a magnetic rack. Heating was performed using a Thermo Veriti 96-well Thermal Cycler PCR instrument. Reverse transcription and quantitative PCR procedures were the same as in 2.1.2 and 2.1.3.

[0175] Example 6: Off-target detection

[0176] RNA sequencing analyzes differential gene expression at the transcriptome level to assess the off-target risk of dsRNA after acting on cells. Hep3B cells and Huh-7 cells were cultured in a medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and double-antibody (Gibco) at 5% CO2 and 37°C. After the cells grew to almost completely cover the culture flask, they were digested with trypsin and resuspended. The resuspended cells were adjusted to a density of 5×10 6 Cells were inoculated into T75 culture flasks and suspended with dsRNA transfection complexes. The dsRNA transfection complex was obtained by mixing 60 μL / bottle of liposome RNAiMax (Thermo) in Opti-MEM (Gibco) and dsRNA in a 1:1 ratio. After a certain period of cell culture, the cells were digested with trypsin and resuspended, washed once with PBS (Gibco), centrifuged to remove the supernatant, and fully lysed with 1 mL of TRNzol (TIANGEN). The samples were frozen at -80°C. Subsequent RNA extraction and transcriptome sequencing were performed by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0177] Example 7: In vivo efficacy of C5 dsRNA in mice

[0178] To evaluate the effect of dsRNA on the in vivo activity of C5, in vivo activity assays were performed using male humanized C5 mice. Pre-dose serum samples were obtained on day -1. The dsRNA conjugates were diluted in saline and administered subcutaneously on day 1 according to the experimental protocol. A blank saline solution was used as a negative control. Plasma was collected on days 1, 3, 7, 14, 21, 28, 35, 42, and 49. Plasma C5 and C5a protein levels were measured by ELISA according to the supplier's protocol (C5: abcam, ab125963; C5a: R&D systems, DY2037). Specifically, the coating antigen was diluted to 5 μg / ml in coating buffer, and 50 μl was added to each well of the ELISA plate. The plate was coated overnight at 4°C. The coating buffer was discarded (patted dry and then blotted with lint-free paper), and the plate was washed with 1× PBST, pH 7.2-7.4. Add 250 μl of blocking solution (1% BSA) to each well and block at 37°C for 2 hours. Wash the plate three times with 1×PBST washing solution, each time for 3 minutes to 5 minutes. Dilute the test serum with 1×PBST buffer, add 100 μl to each well after dilution, and incubate at 37°C for 1.5 hours. After washing the plate, add 50 μl of enzyme-labeled secondary antibody HRP diluted in 1×PBST buffer to each well and incubate at 37°C for 1 hour. After washing the plate, add 50 μl of substrate solution (TMB) to each well and let it develop at room temperature in the dark for 10 minutes. After color development, add 50 μl of stop solution (2 mol / L H2SO4) to each well to terminate the reaction. Use a microplate reader to read the OD value at 450 nm and determine the serum C5 and C5a levels using the standard curve.

[0179] C5 protein levels were normalized for each animal. Normalization was performed by dividing the C5 protein level for each animal at a time point by the animal's pre-treatment expression level (in this case, day -1) to determine a "normalized to pre-treatment" ratio. Expression at a specific time point was then normalized to the saline group by dividing the "normalized to pre-treatment" ratio for the individual animal by the average "normalized to pre-treatment" ratio for all mice in the saline group.

[0180] The results are shown in Figures 9 to 16.

[0181] Example 8: In vivo efficacy of C5 dsRNA in healthy cynomolgus monkeys

[0182] To evaluate the in vivo activity of dsRNA against C5, in vivo activity assays were performed in healthy cynomolgus monkeys. Pre-dose serum samples were obtained on days -7 and -14. The dsRNA conjugate was diluted in saline and subcutaneously injected on day 1 at doses of 5 mg / kg and 25 mg / kg according to the experimental design. A blank saline solution was used as a negative control. Serum was collected on days 3, 7, 14, 28, 42, 56, 70, and 84. C5 protein levels in serum were measured by ELISA according to the protocol provided by the supplier (abcam, ab125963). MBL pathway activity, classical pathway activity, and alternative pathway activity in serum were measured by ELISA according to the protocol provided by the supplier (WIESLAB Complement System).

[0183] C5 protein levels, MBL pathway activity, classical pathway activity, and alternative pathway activity were normalized for each animal. For normalization, the C5 protein level, MBL pathway activity, classical pathway activity, and alternative pathway activity for each animal at a time point were divided by the animal's pre-treatment expression level or activity (the average of the pre-experimental samples from that group) to determine a "normalized to pre-treatment" expression ratio or activity ratio. Expression at a specific time point was then normalized to the saline group by dividing the "normalized to pre-treatment" ratio for the individual animal by the average "normalized to pre-treatment" ratio for all mice in the saline group.

[0184] The test results are shown in Figures 20 and 21, where the data for cemdisiran are from the literature: Pharmacokinetics and pharmacodynamics of pozelimab alone or in combination with cemdisiran in non-human primates (Devalaraja-Narashimha K, Huang C, Cao M, Chen YP, Borodovsky A, Olson WC, et al. (2022) PLoS ONE 17(6): e0269749). It can be seen that at equal doses, C5-210 is superior to cemdisiran in terms of PD marker C5 serum protein concentration and classical pathway activity inhibition.

[0185] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all fall within the scope defined by the claims of the present invention.

Claims

1. A dsRNA comprising a sense strand and an antisense strand, the antisense strand comprising at least 15, such as at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21, consecutive nucleotides that differ from any one of the sequences shown in Table 2 by 0, 1, 2, or 3 nucleotides, and the sense strand having at least 15 nucleotides that are complementary to the antisense strand.

2. The dsRNA according to claim 1, wherein The sense strand sequence is at least substantially complementary to the antisense strand sequence, and preferably is completely complementary.

3. The dsRNA according to claim 1 or 2, wherein The length of the sense strand and the antisense strand are each independently 17 to 25, preferably 19 to 23, more preferably 19 to 21 nucleotides.

4. The dsRNA according to any one of claims 1 to 3, wherein The dsRNA comprises any one of the antisense or sense strand sequences in Table 2, preferably any one of the sense or antisense strands shown in SEQ ID NOs: 223-478 or SEQ ID NOs: 513-520.

5. The dsRNA according to any one of claims 1 to 4, wherein The dsRNA comprises the antisense strand sequence and the sense strand sequence shown in the duplex sequence in Table 2.

6. The dsRNA according to any one of claims 1 to 5, wherein The antisense strand comprises completely consecutive nucleotides selected from SEQ ID NO: 224, 226, 228, 230, 232, 234, 236, 238, 256, 258, 260, 262, 272, 274, 276, 278, 448, 450, 452, 454 or 456.

7. The dsRNA according to any one of claims 1 to 6, wherein The sense strand comprises completely consecutive nucleotides selected from SEQ ID NO: 223, 225, 227, 229, 231, 233, 235, 237, 255, 257, 259, 261, 271, 273, 275, 277, 447, 449, 451, 453 or 455.

8. The dsRNA according to any one of claims 1 to 7, wherein The dsRNA includes a duplex selected from C5-112 to C5-209 and C5-215 to C5-239; in particular, it includes a duplex selected from C5-112, C5-113, C5-114, C5-115, C5-116, C5-117, C5-118, C5-119, C5-128, C5-129, C5-130, C5-131, C5-136, C5-137, C5-138, C5-139, C5-224, C5-225, C5-226, C5-227, and C5-228.

9. A double-stranded RNAi agent comprising the dsRNA of any one of claims 1 to 8, and optionally comprising a targeting ligand.

10. The double-stranded RNAi agent according to claim 9, wherein The targeting ligand comprises N-acetyl-galactosamine (GalNAc) or a derivative thereof.

11. The double-stranded RNAi agent according to claim 9 or 10, wherein The structure of the double-stranded RNAi agent is selected from the following Formula 1 or Formula 2: where R 2 The dsRNA according to any one of claims 1 to 8, wherein R 2 Conjugating to a targeting ligand through the 3' end or 5' end of the sense strand, preferably through the 3' end (eg, phosphate group) of the sense strand to form a double-stranded RNAi agent; Preferably, the double-stranded RNAi agent is any one of the double-stranded RNAi agents listed in Table 3 or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition, characterized in that It comprises the double-stranded RNAi agent according to any one of claims 9 to 11 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, wherein The pharmaceutical composition is formulated for administration by injection or infusion.

14. A method for inhibiting C5 expression in a cell, comprising: The cell is contacted with the double-stranded RNAi agent according to any one of claims 9 to 11 or the pharmaceutical composition according to any one of claims 12 to 13.

15. The method according to claim 14, wherein The cells are hepatocytes or adipocytes.

16. Use of the double-stranded RNAi agent of any one of claims 9 to 11 or the pharmaceutical composition of any one of claims 12 to 13 in the preparation of a medicament for treating a disease or condition mediated by C5 expression.

17. The use according to claim 16, wherein The disease or condition mediated by C5 expression is selected from the group consisting of paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, typical hemolytic uremic syndrome, hemolytic uremic syndrome associated with Shiga toxin-producing Escherichia coli, myasthenia gravis, rheumatoid arthritis, Goodpasture's syndrome, glomerulonephritis (e.g., IgA nephropathy, membranous nephropathy, antineutrophil cytoplasmic antibody-associated vasculitis, lupus nephritis, membranoproliferative nephritis), Hashimoto's thyroiditis, ischemia-reperfusion injury, septic shock, neuromyelitis optica, antibody-mediated renal transplant rejection, Guillain-Barré syndrome, Degos' syndrome, diabetic angiopathy, amyotrophic lateral sclerosis, dense deposit disease, Parkinson's disease, autoimmune encephalitis, IgG4-related disease, asthma, antiphospholipid antibody syndrome, ischemia-reperfusion injury, multifocal motor neuropathy, multiple sclerosis, thrombotic thrombocytopenic purpura, spontaneous abortion, habitual abortion, traumatic brain injury, cold agglutinin disease, dermatomyositis, graft dysfunction, myocardial infarction, sepsis, atherosclerosis, septic shock, spinal cord injury, psoriasis, macular degeneration, autoimmune hemolytic anemia, antiphospholipid syndrome, myocarditis, immune complex vasculitis, Takayasu's disease and Kawasaki disease; in particular, selected from paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, IgA nephropathy, rheumatoid arthritis or myasthenia gravis.

18. The use according to claim 16 or 17, wherein The drug is administered simultaneously or sequentially with the additional therapeutic agent.

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