Compositions and methods for inhibiting complement component C3 protein expression

Abstract

The present invention provides compositions and methods useful for inhibiting complement component C3 gene expression and for treating C3 related diseases and disorders. In particular, the present invention provides dsRNA agents, antisense polynucleotide agents, compositions comprising dsRNA agents, and compositions comprising antisense polynucleotide agents that can be used to reduce C3 gene expression in cells and subjects.

Description

Compositions and methods for inhibiting complement component C3 protein expression Technical Field

[0001] Some embodiments of the present invention relate to compositions and methods that can be used to inhibit the expression of complement component C3 protein. Background Art

[0002] First discovered in 1890, complement is part of the innate immune system that protects the host from invading pathogens and serves as the first line of defense against foreign pathogens. Activation of complement leads to a series of sequential enzymatic reactions, known as complement activation, which results in the formation of the potent anaphylatoxins C3a and C5a, triggering a plethora of physiological responses ranging from chemoattraction to cell apoptosis. Complement was initially thought to play a key role in innate immunity against invading pathogens. However, recent evidence has revealed that complement also plays a crucial role in adaptive immunity involving T and B cells, contributing to pathogen elimination (Dunkelberger JR and Song WC. (2010) Cell Res. 20:34; Molina H, et al. (1996) Proc Natl Acad Sci US A. 93:3357), maintaining immunogenicity to prevent viral reinvasion, and various human pathological conditions (Qu, H, et al. (2009) Mol Immunol. 47:185; Wagner, E. and Frank MM. (2010) No / Rev Drug Discov. 9:43). The complement system is composed primarily of proteins, including complement component protein C3 (also referred to herein as C3). These proteins are synthesized in large quantities and secreted into the blood by hepatocytes in the liver, activating the system's inflammatory response, leading to phagocyte attraction and opsonization, thereby clearing pathogens, immune complexes, and cellular debris.

[0003] Complement component C3 is a key component of the complement system activation pathway. Complement activation is known to occur through three different pathways: the alternative pathway, the classical pathway, and the agglutinin pathway, all of which converge in the formation of the so-called complement component C3 convertase complex. These enzyme complexes cleave the complement component C3 protein into C3a and C3b. Once cleaved, C3b forms part of a complex that, in turn, cleaves C5 into C5a and C5b. After cleavage, C5b is one of the key components of the major complement pathway effector, the membrane attack complex. Therefore, the role of complement component C3 (also known as C3) is essential, as it plays a role in all three pathways of complement activation.

[0004] Abnormal acquisition or genetic activation of the complement system and abnormal or overexpression of C3 cause many different diseases, including, for example: kidney-related C3 glomerulopathy (C3G), immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus, lupus nephritis, ischemia / reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and diseases of other organs associated with C3, such as neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), primary membranous nephropathy, and diseases of other organs known to be associated with complement dysfunction, such as age-related macular degeneration (AMD), rheumatoid arthritis (RA), anti-neutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegenerative diseases.

[0005] Currently, there are treatments available to treat complement component C3-related diseases, but these diseases require time-consuming and invasive management, are costly, and represent a significant unmet clinical need; therefore, there is a need in the art for alternative or combination therapies for patients suffering from complement component C3-related diseases.

[0006] Summary of the Invention

[0007] According to one aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C3 is provided, the dsRNA agent comprising a sense strand and an antisense strand, comprising a region complementary to the C3 RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in Tables 1-3 by 0, 1, 2 or 3 nucleotides, and optionally comprises a targeting ligand.

[0008] In some embodiments, the region complementary to the C3 RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in Tables 1-3.

[0009] In some embodiments, the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1 and provided in one of Tables 1-3.

[0010] In some embodiments, the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO: 1 and provided in one of Tables 1-3.

[0011] In some embodiments, the dsRNA agent comprises any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent.

[0012] In some embodiments, the dsRNA agent comprises any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.

[0013] In some embodiments, the dsRNA agent comprises any one of the antisense strand sequences listed in Tables 1-3.

[0014] In some embodiments, the dsRNA agent comprises any one of the sequences listed as duplex sequences in Tables 1-3.

[0015] In some embodiments, the dsRNA agent comprises at least one modified nucleotide.

[0016] In some embodiments, all or substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least one modified nucleotide comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seco nucleotide mimetics, locked nucleotides, unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides comprising a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or an unnatural base comprising nucleotide. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein less than 6 2'-fluoro nucleotide modified nucleotides. In certain embodiments, the antisense strand comprises 3 or 5 2'-fluoro nucleotides, preferably, the antisense strand comprises 5 2'-fluoro nucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein less than 4 2'-fluoro nucleotide modified nucleotides. In certain embodiments, the sense strand comprises 3 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least 16 modified nucleotides are 2'-O-methyl nucleotides and are positioned at the 2nd, 7th, 12th, 14th and / or 16th positions of the 5' end of the antisense strand are 2'-fluoro nucleotide modified nucleotides (calculated from the nucleotide of the first paired 5' of the antisense strand). In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least 18 of the modified nucleotides are 2'-O-methyl nucleotides and the 9th, 11th, and / or 13th positions located at the 3' end of the sense strand are 2'-fluoro modified nucleotides (calculated from the first paired nucleotide at the 3' end of the sense strand). In some embodiments, the antisense strand comprises, in the direction from the 5' end to the 3' end, nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand are 2'-fluoro modified nucleotides, calculated from the first paired nucleotide at the 5' end of the antisense strand, and the nucleotides at other positions in each antisense strand are independently non-fluoro modified nucleotides.In some embodiments, the antisense strand comprises positions 2, 5, 12, 14, and 16 of the antisense strand in the direction from the 5' end to the 3' end, which are 2'-fluorine-modified nucleotides, calculated from the first paired nucleotide of the 5' end of the antisense strand, and each nucleotide at other positions in the antisense strand is independently a non-fluorine-modified nucleotide. In some embodiments, the sense strand comprises positions 9, 11, and 13 of the sense strand in the direction from the 3' end to the 5' end, which are 2'-fluorine-modified nucleotides, calculated from the first paired nucleotide of the 3' end of the sense strand, and each nucleotide at other positions in the sense strand is independently a non-fluorine-modified nucleotide. In some embodiments, the antisense strand comprises positions 7 of the antisense strand in the direction from the 5' end to the 3' end, which are UNA-modified nucleotides, calculated from the first paired nucleotide of the 5' end of the antisense strand.

[0017] In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C3 comprises a sense strand and an antisense strand, wherein nucleotide positions 2 to 18 in the antisense strand comprise a region complementary to the C3 RNA transcript, the antisense strand is fully or partially complementary to the sense strand, and optionally comprises a targeting ligand, wherein each strand is 17 to 40 nucleotides in length, wherein the sense strand sequence comprises the formula (I): 5′-(N′ L ) n′ N′ L N′ L N′ L N′ L N′ L N′ F N′ L N′ F N′ L N′ F N′ L N′ L N′ L N′ L N′ L N′ L N′ L (N′ L ) m′ -3′ (I)

[0018] Where: Each N' F represents a 2'-fluoro modified nucleotide; each N' L independently represents a modified or unmodified nucleotide but does not represent a 2'-fluoro modified nucleotide, n' is an integer from 0 to 7, and m' is an integer from 0 to 3. In certain embodiments, m' is 1. In certain embodiments, n' is 3 and m' is 1; or n' is 0 and m' is 0; or n' is 3 and m' is 3.

[0019] In some embodiments, the dsRNA agent includes an E-vinylphosphonate nucleotide at the 5' end of the guide strand.

[0020] In some embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

[0021] In some embodiments, all or substantially all nucleotides of the sense and antisense strands are modified nucleotides. In some embodiments, the modified sense strand is a modified sense strand sequence listed in Table 2 and / or Table 3. In some embodiments, the modified antisense strand is a modified antisense strand sequence listed in Table 2 and / or Table 3.

[0022] In some embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is between 16 and 23 nucleotides. In some embodiments, the length of the complementary region is 19-21 nucleotides. In some embodiments, the length of the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of each strand in the dsRNA agent does not exceed 40 nucleotides. In some embodiments, the length of each strand does not exceed 30 nucleotides. In some embodiments, the length of each strand does not exceed 25 nucleotides. In some embodiments, the length of each strand does not exceed 23 nucleotides. In some embodiments, the length of each strand does not exceed 21 nucleotides. In some embodiments, the length of each strand is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides.

[0023] In some embodiments, the C3 RNA transcript is SEQ ID NO: 1.

[0024] In some embodiments, a dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups.

[0025] In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc).

[0026] In some embodiments, the targeting group or linking group has a structure as shown in formula (X), comprising a targeting moiety, a linker, and a linker W, wherein the targeting moiety is selected from an N-acetyl-galactosamine derivative (GalNAc), which is linked to the linker W via a linker, and the linker W has a structure as shown in formula (XI), X is selected from O, NH2 or S, and Y ﹣ Selected from: O ﹣ 、S ﹣ , methyl or NRaRb, Ra and Rb are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, or Ra and Rb together with the attached atoms are linked to form a 3-12 membered heterocycloalkyl containing 1-3 N, O, S heteroatoms,

[0027] In some preferred embodiments, in the substituted or unsubstituted C1-C6 alkyl group, or the substituted or unsubstituted C3-C6 cycloalkyl group, the substituent is selected from hydroxyl group and amino group. In some embodiments, X is selected from O or S, Y ﹣ Selected from: O ﹣ 、S ﹣ In some embodiments, the linker in the targeting group is selected from polyethylene glycol, optionally substituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 In some embodiments, the substituted or unsubstituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 In the amide, the substituent is selected from hydroxyl and carbonyl.

[0028] In some embodiments, the linker in the targeting group is preferably selected from the following fragments:

[0029] Each m is an integer from 1 to 6, each n, o, and p is 0 or 1, and each q is an integer from 1 to 6. 1 With q 2 Each independently is 0, 1 or 2.

[0030] In some embodiments, the linker in the targeting group is more preferably selected from the following fragments:

[0031] In some embodiments, the targeting moiety in the targeting group has the following structural fragment,

[0032] n" is independently 1 or 2.

[0033] In some embodiments, the targeting group has the following structure:

[0034] In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 5'-end of the sense strand. In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 3'-end of the sense strand.

[0035] In some embodiments, the antisense strand comprises an inverted abasic residue at the 3'-terminus. In some embodiments, the sense strand comprises one or two inverted abasic residues at the 3' or / and 5' termini.

[0036] In some embodiments, the dsRNA agent has two blunt ends.

[0037] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide in length. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides in length. In one embodiment, the sense strand has an overhang of 1-10 nucleotides at the 3'-end and / or the 5'-end, such as an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0038] Another aspect of the present invention provides a double-stranded ribonucleic acid (dsRNA) agent expressed by complement component C3, the dsRNA agent comprising a sense strand and an antisense strand, wherein each strand is 14 to 30 nucleotides in length, comprising a region complementary to a C3 RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in Formula I by 0, 1, 2, or 3 nucleotides, and optionally comprises a targeting ligand:

[0039] Double-stranded RNA (dsRNA) reagent antisense strand: 5'-N a -(A1 A2 A3 A4)iN b –(B1 B2 B3 B4)-N b -(C1 C2 C3 C4 C5)jN a -3'

[0040] Wherein: i and j are independently selected from 0 or 1;

[0041] Each N aand N b Each independently represents 0-17 oligonucleotides;

[0042] A1 A2 A3 A4 represents a motif of four consecutive nucleotides modified by lowercase 2'-OMe, uppercase 2'-fluorine, lowercase 2'-OMe, and lowercase 2'-OMe, respectively. Preferably, A1 and A2, and A2 and A3 further contain phosphorothioate internucleoside linkages.

[0043] B1 B2 B3 B4 represents a motif in which four consecutive nucleotides are modified with lowercase 2'-OMe;

[0044] C1 C2 C3 C4 C5 represents a motif in which five consecutive nucleotides are modified with lowercase 2'-OMe, preferably further comprising phosphorothioate internucleoside linkages between C3 and C4 and between C4 and C5.

[0045] In some embodiments, N a Each independently represents 0 oligonucleotide, N b Each independently represents 2-5 chemically modified oligonucleotides used herein, and i and j each represent 1.

[0046] In some embodiments, the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1. In some embodiments, the antisense strand of the dsRNA is fully complementary to any target region of SEQ ID NO: 1. In some embodiments, the sense strand sequence of any of the dsRNAs is at least substantially complementary to the antisense strand sequence of the dsRNA agent. In some embodiments, the sense strand sequence of any of the dsRNAs is fully complementary to the antisense strand sequence of the dsRNA agent.

[0047] In some embodiments, all or substantially all nucleotides of the sense strand are modified nucleotides.

[0048] In some embodiments, the sense strand comprises one or two inverted abasic residues at the 3' or / and 5' termini.

[0049] In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide in length. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides in length. In one embodiment, the sense strand has a 1-10 nucleotide overhang at the 3'-end and / or the 5'-end, such as an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0050] In some embodiments, the dsRNA agent comprises at least one modified nucleotide and also comprises one or more targeting groups or linking groups. In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand. In some embodiments, the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc). In some embodiments, the targeting group is as used herein, any of GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16.

[0051] According to another aspect of the present invention, a composition is provided, comprising any embodiment of the above-mentioned dsRNA agent aspect of the present invention.

[0052] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.In some embodiments, the composition further comprises one or more additional therapeutic agents.

[0053] In certain embodiments, the composition is packaged in a kit, container, package, dispenser, prefilled syringe, or vial.

[0054] In some embodiments, the composition is formulated for subcutaneous administration or is formulated for intravenous (IV) administration.

[0055] According to another aspect of the present invention, a cell is provided, comprising any embodiment of the above-mentioned dsRNA agent of the present invention.

[0056] In some embodiments, the cell is a mammalian cell, optionally a human cell.

[0057] According to another aspect of the present invention, a method for inhibiting C3 gene expression in a cell is provided, the method comprising: (i) preparing a cell comprising an effective amount of the above dsRNA agent or any embodiment of the above composition.

[0058] In certain embodiments, the method further comprises: (ii) maintaining the prepared cells for a sufficient period of time to achieve degradation of the mRNA transcript of the C3 gene, thereby inhibiting expression of the C3 gene in the cells.

[0059] In some embodiments, the cells are in a subject and the dsRNA agent is administered subcutaneously to the subject.

[0060] In some embodiments, the cells are in a subject and the dsRNA agent is administered to the subject by IV administration.

[0061] In certain embodiments, the method further comprises assessing inhibition of the C3 gene after administering the dsRNA agent to the subject, wherein the means for assessing comprises: (i) determining one or more physiological characteristics of a C3-related disease or condition in the subject, and (ii) comparing the determined physiological characteristics to a baseline pre-treatment physiological characteristic of the C3-related disease or condition and / or a control physiological characteristic of the C3-related disease or condition, wherein the comparison indicates the presence or absence of inhibition of C3 gene expression in the subject.

[0062] In some embodiments, the physiological characteristic determined is the level of C3 in the blood;

[0063] In some embodiments, for example, paroxysmal nocturnal hemoglobinuria (PNH), a relatively rare disease that includes an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis, hemoglobinuria, bone marrow failure, and thrombophilia (tendency to form blood clots), a decrease in C3 levels in the blood and / or C3 indicates a decrease in C3 gene expression in the subject.

[0064] According to another aspect of the present invention, a method for inhibiting C3 gene expression in a subject is provided, comprising administering to the subject an effective amount of the aforementioned dsRNA agent embodiment or the aforementioned composition embodiment.

[0065] In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In certain embodiments, the dsRNA agent is administered to the subject by IV administration. In some embodiments, the method further comprises: evaluating the inhibition of the C3 gene after administration of the dsRNA agent, wherein the evaluation means comprises: (i) determining one or more physiological characteristics of the C3-related disease or condition of the subject; (ii) comparing the determined physiological characteristics with the baseline pre-treatment physiological characteristics of the C3-related disease or condition and / or the control physiological characteristics of the C3-related disease or condition; wherein the comparison result indicates the presence or absence of inhibition of C3 gene expression in the subject. In some embodiments, the physiological characteristic determined is the level of C3 in the blood; in some embodiments, paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disease that includes an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis, hemoglobinuria, bone marrow failure, and thrombophilia (tendency to form blood clots) The physiological characteristics determined. A decrease in C3 levels and / or C3 in the blood indicates a decrease in C3 gene expression in the subject.

[0066] According to another aspect of the present invention, a method for treating a disease or condition associated with C3 protein is provided, comprising: administering to a subject an effective amount of any embodiment of the aforementioned dsRNA agent of the present invention or any embodiment of the aforementioned composition of the present invention to inhibit C3 gene expression.

[0067] In certain embodiments, the C3-related disorder is selected from the group consisting of: C3 glomerulopathy (C3G) associated with the kidney, immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus, lupus nephritis, ischemia / reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and diseases of other organs associated with C3, such as neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis, and leukemia. None (MG), primary membranous nephropathy, diseases of other organs also known to be associated with complement dysfunction, such as age-related macular degeneration (AMD), rheumatoid arthritis (RA), anti-neutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegenerative diseases, preferably C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), lupus nephritis, IgA nephropathy (IgAN) and primary membranous nephropathy, age-related macular degeneration (AMD).

[0068] In some embodiments, the method further comprises administering to the subject an additional therapeutic regimen.

[0069] In some embodiments, the additional treatment regimen includes treatment of a C3-related disease or condition. In certain embodiments, the additional treatment regimen includes: administering one or more C3 antisense polynucleotides of the present invention to a subject; administering a non-C3 dsRNA therapeutic to a subject; and performing a behavioral change in the subject. In some embodiments, the non-C3 dsRNA therapeutic is one of the following: an additional therapeutic agent, such as an anti-complement component C5 antibody or antigen-binding fragment thereof (e.g., eculizumab), an iRNA targeting complement component C5, and / or a C3 peptide inhibitor (e.g., comprusinvastatin), thereby treating a subject suffering from a disease that would benefit from reduced C3 expression.

[0070] In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In certain embodiments, the dsRNA agent is administered to the subject by IV administration. In some embodiments, the method further comprises determining the efficacy of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject. In some embodiments, the means for determining the efficacy of the treatment in the subject comprises: (i) determining one or more physiological characteristics of a C3-related disease or condition in the subject; (ii) comparing the determined physiological characteristics with the baseline pre-treatment physiological characteristics of the C3-related disease or condition, wherein the comparison result indicates one or more of the presence, absence, and level of efficacy of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject. In some embodiments, the physiological characteristic determined is the level of C3 in the blood; in some embodiments, paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disease that includes physiological characteristics determined by an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis, hemoglobinuria, bone marrow failure, and thrombophilia (tendency to form blood clots). A decrease in C3 levels and / or hemolysis in the blood indicates the presence of effectiveness of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject.

[0071] According to another aspect of the present invention, a method for reducing the level of C3 protein in a subject compared to the baseline pre-treatment level of C3 protein in the subject is provided, comprising administering to the subject an effective amount of any embodiment of the aforementioned dsRNA agent of the present invention or any embodiment of the aforementioned composition of the present invention to reduce the level of C3 gene expression. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or by IV.

[0072] According to another aspect of the present invention, a method is provided for altering a physiological characteristic of a C3-related disease or condition in a subject compared to a baseline pre-treatment physiological characteristic of the C3-related disease or condition in the subject, the method comprising administering to the subject an effective amount of any embodiment of the aforementioned dsRNA agent aspects of the present invention or any embodiment of the aforementioned composition of the present invention to alter the physiological characteristic of the C3-related disease or condition in the subject. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or by IV. In certain embodiments, the physiological characteristic is the level of C3 in the blood; in some embodiments, the physiological characteristic determined is a decrease in hemolysis.

[0073] Sequence Description

[0074] Duplexes AV00375 to AV00447 are shown in Table 1 and their sense strand sequences are shown.

[0075] Duplexes AV00375 to AV00447 are shown in Table 1 and their antisense strand sequences are shown.

[0076] SEQ ID NO: 1 is human C3 mRNA [NCBI Reference Sequence: NM_000064.4].

[0077] In the sequences shown in Table 2, chemical modifications are indicated as follows: upper case: 2'-fluoro; lower case: 2'-OMe; phosphorothioate: *; UNA: open nucleic acid.

[0078] In the sequences shown in Table 3, the delivery molecules used in the in vivo studies are indicated as "GLX-n" at the 3' or 5' end of each sense strand. Chemical modifications are indicated as follows: uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *; UNA: open nucleic acid; Invab = inverted abasic. DETAILED DESCRIPTION

[0079] Some embodiments of the present invention include RNAi agents capable of inhibiting the expression of the complement component C3 gene, such as, but not limited to, double-stranded (ds) RNAi agents. The C3 dsRNA agents of the present invention can target C3 transcripts, resulting in inhibition of C3 protein expression. Some embodiments of the present invention also include compositions comprising C3 RNAi agents and methods of using the compositions. The C3 RNAi agents disclosed herein can be attached to a delivery compound for delivery to cells, including hepatocytes. The pharmaceutical compositions of the present invention may include at least one C3 dsRNA agent and a delivery compound. In some embodiments of the present invention, the delivery compound is a GalNAc-containing delivery compound. The C3 RNAi agent delivered to the cell can inhibit C3 gene expression, thereby reducing the activity of the gene's C3 protein product in the cell. The dsRNAi agents of the present invention can be used to treat C3-related diseases and conditions. Such dsRNAi agents include, for example, duplexes AV00375 to AV00447 shown in Table 1. In some embodiments, preferred dsRNAi agents include, for example, duplexes AV00378, AV00383, AV00385, AV00386, AV00388, AV00375, AV00413, AV00416, or AV00429. In other embodiments, preferred dsRNAi agents of Table 2 include, for example, duplexes AV00378, AV00383, AV00385, AV00386, AV00388, AV00375, AV00413, AV00416, or AV00429.

[0080] In some embodiments of the present invention, reducing C3 expression in a cell or subject is used to treat a disease or condition associated with C3 expression in a cell or subject, respectively. Non-limiting examples of diseases and conditions that can be treated by reducing C3 activity are: C3 glomerulopathy (C3G) associated with the kidney, immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus, lupus nephritis, ischemia / reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and diseases of other organs associated with C3, such as neuromyelitis optica (NMO), multifocal motor neuropathy (MMN). , myasthenia gravis (MG), primary membranous nephropathy, and diseases of other organs known to be associated with complement dysfunction, such as age-related macular degeneration (AMD), rheumatoid arthritis (RA), anti-neutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegenerative diseases, preferably C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), lupus nephritis, IgA nephropathy (IgAN) and primary membranous nephropathy, age-related macular degeneration (AMD).

[0081] Described below are compositions comprising single-stranded (ssRNA) and double-stranded (dsRNA) C3 agents to inhibit C3 gene expression, as well as compositions and methods for treating diseases and conditions caused or regulated by C3 gene expression. As used herein, diseases and / or conditions caused or regulated by the presence and / or level of C3 gene expression are referred to as "C3-related diseases and / or conditions." The term "RNAi" is also known in the art and may be referred to as "siRNA."

[0082] As used herein, the term "RNAi" refers to an agent comprising RNA and mediating the targeted cutting of RNA transcripts by RNA-induced silencing complex (RISC) pathways. As known in the art, the RNAi target region refers to a continuous portion of the nucleotide sequence of the RNA molecule formed during gene transcription, including messenger RNA (mRNA), which is a processed product of primary transcript RNA. The target portion of the sequence will be at least long enough to be used as a substrate for RNAi directed cutting at or near this portion. The target sequence can be 8-30 nucleotides long (including end values), 10-30 nucleotides long (including end values), 12-25 nucleotides long (including end values), 15-23 nucleotides long (including end values), 16-23 nucleotides long (including end values), or 18-23 nucleotides long (including end values), and includes all shorter lengths within each specified range. In some embodiments of the present invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides long. In certain embodiments, the target sequence is between 9 and 26 nucleotides in length (inclusive), including all subranges and integers therebetween. For example, although not intended to be limiting, in certain embodiments of the present invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is completely or at least substantially complementary to at least a portion of the RNA transcript of the C3 gene.

[0083] Some aspects of the invention include pharmaceutical compositions comprising one or more C3 dsRNA agents and a pharmaceutically acceptable carrier.In certain embodiments of the invention, the C3 RNAi as described herein inhibits the expression of C3 protein.

[0084] In some embodiments of the present invention, the pharmaceutical composition comprises one, two, three, or more independent anti-C3 dsRNA agents and may further comprise one or more independently selected delivery compounds. In some embodiments, two, three, four, or more anti-C3 dsRNAs that are capable of targeting one, two, three, four, or more different locations / regions of C3 mRNA are administered to a cell or subject.

[0085] As used herein, "dsRNA agent" refers to a composition comprising RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that are capable of degrading or inhibiting the translation of a target mRNA transcript. Although not wishing to be limited to a particular theory, the dsRNA agents of the present invention may act via an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)), or by any alternative mechanism or pathway. Methods for achieving gene silencing in plant, invertebrate, and vertebrate cells are well known in the art (see, e.g., Sharp et al., Genes Dev. 2001, 15:485; Bernstein, et al., (2001) Nature 409:363; Nykanen, et al., (2001) Cell 107:309; and Elbashir, et al., (2001) Genes Dev. 15:188), the disclosures of each of which are incorporated herein by reference in their entirety. Gene silencing approaches known in the art can be used in conjunction with the disclosure provided herein to achieve inhibition of C3 expression.

[0086] The dsRNA agents disclosed herein are composed of a sense strand and an antisense strand, and include, but are not limited to, short interfering RNA (siRNA), RNAi agents, microRNA (miRNA), short hairpin RNA (shRNA), and Dicer substrates. The antisense strand of the dsRNA agents described herein is at least partially complementary to the targeted mRNA. It is understood in the art that dsRNA duplex structures of various lengths can be used to inhibit target gene expression. For example, dsRNAs having duplex structures of 19, 20, 21, 22, and 23 base pairs are known to be effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). Shorter or longer RNA duplex structures are also known in the art to be effective in inducing RNA interference. The C3 dsRNA in certain embodiments of the present invention may comprise at least one strand of at least 21 nt in length, or the duplex may have a length based on one of the sequences listed in Tables 1-4 minus 1, 2, 3 nt, or less. Compared to the dsRNAs listed in Tables 1-3, respectively, a reduction of 4 nucleotides at one or both ends can also be effective. In some embodiments of the present invention, C3 dsRNA agents may have a partial sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one or more sequences in Tables 1-3, and their ability to inhibit C3 gene expression differs by no more than 5%, 10%, 15%, 20%, 25% or 30% from the level of inhibition produced by a dsRNA comprising the full sequence (also referred to herein as the "parent" sequence). The sense sequences, antisense sequences, and duplexes disclosed in Tables 1-3 may be referred to herein as "parent" sequences, meaning that the sequences disclosed in Tables 1-3 can be modified, shortened, extended, include substitutions, etc., as described herein, and the resulting sequences retain all or at least some of the efficacy of their parent sequences in the methods and compositions of the present invention.

[0087] Certain embodiments of the compositions and methods of the present invention include single-stranded RNA in the composition and / or administer single-stranded RNA to a subject. For example, the antisense strands listed in any of Tables 1-3 can be used as a composition or within a composition that, when administered to a subject, reduces C3 polypeptide activity and / or C3 gene expression in the subject. Tables 1-3 show the antisense strand and sense strand core extension base sequences of certain C3 dsRNA agents. Single-stranded antisense molecules that can be included in certain compositions of the present invention and / or administered in certain methods of the present invention are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents." Single-stranded sense molecules that can be included in certain compositions and / or administered in certain methods of the present invention are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents." The term "base sequence" herein refers to a polynucleotide sequence without chemical modifications or delivery compounds. For example, the sense strands shown in Table 1 correspond to the corresponding base sequences in Table 3; however, the corresponding sequences in Table 3 show their respective chemical modifications and delivery compounds. Sequences disclosed herein can be assigned identifiers. For example, a single-stranded sense sequence can be identified by "sense strand SS#"; a single-stranded antisense sequence can be identified by "antisense strand AS#"; and a duplex comprising a sense strand and an antisense strand can be identified by "duplex AD#".

[0088] Table 1 includes a sense strand and an antisense strand, and provides identification numbers for duplexes formed by the sense strand and the antisense strand on the same row in Table 1. In certain embodiments of the present invention, the antisense sequence comprises nucleobase u or nucleobase a at position 1 thereof. In certain embodiments of the present invention, the antisense sequence comprises nucleobase u at position 1 of the antisense sequence. As used herein, the term "matching position" refers in a sense to the position in each strand that "pairs" with each other when the two strands are as a duplex. For example, in a 21 nucleobase sense strand and a 21 nucleobase antisense strand, the nucleobase at position 1 of the sense strand is in a "matching position" with the nucleobase at position 21 of the antisense strand. In another non-limiting example, for a 23 nucleobase sense strand and a 23 nucleobase antisense strand, the nucleobase at position 2 of the sense strand is in a matching position with the nucleobase at position 22 of the antisense strand. In another non-limiting example, in an 18-nucleobase sense strand and an 18-nucleobase antisense strand, nucleobase position 1 of the sense strand is in a matching position with nucleobase position 18 of the antisense strand; and nucleobase position 4 of the sense strand is in a matching position with nucleobase position 15 of the antisense strand. A skilled artisan will understand how to identify matching positions between the sense and antisense strands of duplexes and paired strands.

[0089] The first column in Table 1 represents duplex AV# for duplexes containing both sense and antisense sequences in the same row of the table. For example, Table 1 discloses a duplex designated "Duplex AV00375," which contains corresponding sense and antisense strand sequences. Thus, each row in Table 1 identifies a duplex of the present invention, each containing both sense and antisense sequences displayed in the same row, with the designated identifier for each duplex displayed in the first column of that row.

[0090] In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in Table 1 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises a duplex comprising at least one of the base sequences listed in Table 1 and comprising 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 sequence modifications. In some embodiments of the methods of the present invention, the RNAi agent comprising a polynucleotide sequence shown in Table 1 is further coupled to a delivery molecule, a non-limiting example of which is a delivery compound comprising GalNAc.

[0091] Table 1: Unmodified C3 RNAi agent antisense and sense strand sequences. All sequences are shown in the 5' to 3' direction. Duplex AV# is the number assigned to the duplexes of both strands in the same row of the table. "SEQ ID NO: 2-74" in the third column of Table 1 is the sequence number of the sense sequence shown in the second column, and "SEQ ID NO: 75-147" in the fifth column of Table 1 is the sequence number of the antisense sequence shown in the fourth column.

[0092] Table 2 shows the antisense and sense strand sequences of certain chemically modified C3 RNAi agents of the present invention. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a cell and / or a subject. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to the subject comprises the duplexes indicated in the first column of Table 2 and comprises the sequence modifications shown in the sense and antisense strand sequences in the third and sixth columns of the same row of Table 2, respectively. In some embodiments of the methods of the present invention, the sequences shown in Table 2 can be linked (also referred to herein as "conjugated to") to a compound capable of delivering the RNAi agent to the cells and / or tissues of the subject. A non-limiting example of a delivery compound that can be used in certain embodiments of the present invention is a GalNAc-containing compound. In Table 2, the first column represents the duplex AV# of the base sequence, corresponding to Table 1. For the base sequence indicated by duplex AV#, not only the base sequence contained in the sense and antisense strands is shown, but also the specified chemical modification shown in the same row of Table 2. For example, the first row of Table 1 shows the sense and antisense single-stranded base sequences, which together form a duplex, identified as duplex AV#AV00375; while the duplex AV#AV00375 listed in Table 2, as a duplex, contains the base sequences of AV00375-SS and AV00375-AS, and contains the chemical modifications in the sense and antisense sequences shown in the third and sixth columns, respectively. The "sense strand SS#" in the second column of Table 2 is the designated identifier of the sense sequence (including modifications) shown in the third column of the same row. The "antisense strand AS#" in the fifth column of Table 2 is the designated identifier of the antisense sequence (including modifications) shown in the sixth column. The "SEQ ID NO" in the fourth column of Table 2 is the sequence number of the sense sequence (including modifications) shown in the third column of the same row. The "SEQ ID NO" in the seventh column of Table 2 is the sequence number of the antisense sequence (including modifications) shown in the sixth column.

[0093] Table 3 shows certain chemically modified C3 RNAi agent antisense and sense strand sequences of the present invention. In some embodiments of the present invention, the RNAi agent shown in Table 3 is administered to a cell and / or subject. In some embodiments of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 3 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to the subject comprises a duplex identified in the first column of Table 3 and comprises the sequence modifications and / or delivery compounds shown in the sense and antisense strand sequences in the third and sixth columns of the same row of Table 3, respectively. The sequences are used in certain in vivo testing studies described elsewhere herein. In some embodiments of the present invention, the sequences shown in Table 3 can be linked to (also referred to herein as "conjugated to") a compound for delivery, a non-limiting example of which is a GalNAc-containing compound, i.e., a delivery compound identified as "GLX-n" on the sense strand in the third column of Table 3. Certain embodiments of the delivery compound are identified as "GLS-5," "GLS-15," or "GLX-n" on the sense strand in the third column of Table 3. As used herein and as shown in Table 3, "GLS-5," "GLS-15," and "GLX-n" refer to compounds containing GalNAc. As used herein, "GLX" is used to refer to "GLS" or "GLO" delivery compounds ("X" can be "S" or "O"), and GLX-n can be any GLS or GLO delivery compound that can be attached to the 3'- or 5'-end of an oligonucleotide during synthesis. For example, but not limited to, GLX-13 and GLX-14 can be attached to the 3'-end of an oligonucleotide of the present invention during synthesis; GLX-5 and GLX-15 can be attached to the 5'-end of an oligonucleotide of the present invention during synthesis. In some embodiments, as used herein and as shown in Table 3, "GLX-n" is used to indicate that the attached GalNAc-containing compound is replaced with any one of compounds GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16.In some embodiments, GLX-n represents, for example but not limited to, the GalNAc-containing compounds disclosed in the prior art (Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961) that can be used for attachment, which compounds were used in certain in vivo testing studies described elsewhere herein. Those skilled in the art will be able to prepare and use the dsRNA compounds of the present invention, wherein the attached delivery compounds include, but are not limited to: GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16. The structures of each of these are provided elsewhere herein. The first column of Table 3 provides the duplex AD# assigned to the duplex of the sense and antisense sequences in that row of the table. For example, duplex AD#AD00343 is a duplex consisting of sense strand AD00343-SS and antisense strand AD00343-AS. Each row in Table 3 provides a sense strand and an antisense strand and discloses the duplex consisting of the sense strand and antisense strand shown. The "sense strand SS#" in the second column of Table 3 is the designated identifier of the sense sequence (including modifications) shown in the third column of the same row. The "antisense strand AS#" in the fifth column of Table 3 is the designated identifier of the antisense sequence (including modifications) shown in the sixth column. The identifier of certain linked GalNAc-containing GLO compounds is shown as GLX-n, and it should be understood that another of the GLO-n or GLS-n compounds can replace the compound shown as GLX-n, and the resulting compounds are also included in the embodiments of the methods and / or compositions of the present invention. In Table 3, SEQ ID NO: 294 to SEQ ID NO: 342, SEQ ID NO: 392 to SEQ ID NO: 406 are sense strand sequences, and SEQ ID NO: 343 to SEQ ID NO: 391, SEQ ID NO: 407 to SEQ ID NO: 421 are antisense strand sequences. In Table 3, the delivery molecule is indicated as "GLS-5," "GLS-15," or "GLX-n" at the 3' or 5' end of the sense strands SEQ ID NO: 294 to SEQ ID NO: 342, SEQ ID NO: 392 to SEQ ID NO: 406. Chemical modifications are indicated as follows: uppercase: 2'-fluoro; lowercase: 2'-methoxy; thiophosphate: *; Invab: inverted abasic.

[0094] mismatch

[0095] It is known to those skilled in the art that mismatches are tolerable for dsRNA efficacy, particularly in the case of mismatches in the terminal regions of the dsRNA. Certain mismatches are better tolerated, for example, mismatches with wobble base pairs G:U and A:C are better tolerated for efficacy (Du et al., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21; 33(5): 1671-7. Doi: 10.1093 / nar / gki312. Nucleic Acids Res. 2005; 33(11): 3698). In some embodiments of the methods and compounds of the present invention, the C3 dsRNA agent may contain one or more mismatches with the C3 target sequence. In some embodiments, the C3 dsRNA agent of the present invention does not contain mismatches. In certain embodiments, the C3 dsRNA agent of the present invention contains no more than one mismatch. In some embodiments, the C3 dsRNA agents of the present invention contain no more than 2 mismatches. In certain embodiments, the C3 dsRNA agents of the present invention contain no more than 3 mismatches. In some embodiments of the present invention, the antisense strand of the C3 dsRNA agent contains a mismatch with a C3 target sequence that is not located at the center of the region of complementarity. In some embodiments, the antisense strand of the C3 dsRNA agent contains 1, 2, 3, 4, or more mismatches that are located within the last 5, 4, 3, 2, or 1 nucleotides of either or both of the 5' or 3' ends of the region of complementarity. Methods described herein and / or methods known in the art can be used to determine whether a C3 dsRNA agent containing a mismatch with a C3 target sequence is effective in inhibiting expression of a C3 gene.

[0096] Complementarity

[0097] As used herein, unless otherwise indicated, the term "complementarity" when used to describe the relatedness of a first nucleotide sequence (e.g., a C3 dsRNA agent sense strand or a target C3 mRNA) to a second nucleotide sequence (e.g., a C3 dsRNA agent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize [form inter-base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)] with an oligonucleotide or polynucleotide comprising the second nucleotide sequence and to form a duplex or duplex structure under certain conditions. Other conditions may also apply, such as physiologically relevant conditions that may be encountered in an organism. A skilled artisan will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences based on the ultimate application of the hybridizing nucleotides. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, as long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modification.

[0098] For example, a complementary sequence within a C3 dsRNA as described herein comprises base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to one another. It should be understood that in embodiments where two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches for purposes of complementarity determination herein. For example, a C3 dsRNA agent comprising one oligonucleotide of 19 nucleotides in length and another oligonucleotide of 20 nucleotides in length, wherein the longer oligonucleotide comprises a 19-nucleotide sequence that is fully complementary to the shorter oligonucleotide, may be referred to as "fully complementary" for purposes described herein. Thus, as used herein, "fully complementary" means that all (100%) bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

[0099] As used herein, the term "substantially complementary" means that in a hybridization pair of nucleobase sequences, at least about 85% (but not all) of the bases in the contiguous sequence of a first polynucleotide will hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. If the two sequences contain one or more mismatched base pairs upon hybridization, such as at least 1, 2, 3, 4, or 5 mismatched base pairs, the term "substantially complementary" can be used to refer to a first sequence that forms a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp) relative to the second sequence while retaining the ability to hybridize under conditions most relevant to its ultimate application, for example, inhibition of C3 gene expression through the RISC pathway. The term "partially complementary" can be used herein to refer to a hybridization pair of nucleobase sequences in which at least 75% (but not all) of the bases in the contiguous sequence of a first polynucleotide will hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases in the contiguous sequence of the first polynucleotide will hybridize to the same number of bases in the contiguous sequence of the second polynucleotide.

[0100] The terms "complementary," "fully complementary," "substantially complementary," and "partially complementary" as used herein may refer to base matching between the sense and antisense strands of a C3 dsRNA agent, base matching between the antisense strand of a C3 dsRNA agent and the sequence of a target C3 mRNA, or base matching between a single-stranded antisense oligonucleotide and the target C3 mRNA sequence. It should be understood that the term "antisense strand of a C3 dsRNA agent" can refer to the same sequence as a "C3 antisense polynucleotide agent."

[0101] As used herein, the terms "substantially identical" or "substantial identity" used in reference to nucleic acid sequences refer to nucleic acid sequences comprising sequences having at least about 85% or higher sequence identity, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing the optimal alignment of the two sequences over the comparison window. The percentage is calculated by determining the number of positions at which the same nucleic acid base appears in the two sequences to produce the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window and then multiplying the result by 100 to derive the percentage of sequence identity. The invention disclosed herein includes nucleotide sequences substantially identical to those disclosed herein (e.g., in Tables 1-3). In some embodiments, the nucleotide sequence is identical to a sequence disclosed herein (e.g., in Tables 1-3), or has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0102] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence designated using standard nucleotide nomenclature. As used herein, the term "double-stranded RNA" or "dsRNA" refers to a sequence comprising an RNA molecule or RNAi molecule complex having a hybridized double-stranded region comprising two antiparallel and substantially or completely complementary nucleic acid strands, which are respectively referred to as having "sense" and "antisense" orientations relative to the target C3 RNA. The double-stranded region can have any desired length that allows for specific degradation of the target C3 RNA by the RISC pathway, but is typically 9 to 30 base pairs in length, for example 15-30 base pairs in length. Contemplating duplexes between 9 and 30 base pairs, the duplexes can be any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, and any subranges therein, including but not limited to 15-30 base pairs, 15-26 base pairs; 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-27 base pairs, 18-30 base pairs, 18-28 base pairs, 18-30 base pairs, 18-26 base pairs, 18-27 base pairs, 18-28 base pairs, 18-29 base pairs, 18-30 base pairs, 18-29 base pairs, 18-30 base pairs, 18-26 .... -23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs or 21-22 base pairs. The length of C3 dsRNA agents produced in cells by processing with Dicer and similar enzymes is generally in the range of 19-22 base pairs. One strand of the double-stranded region of the C3 dsDNA agent comprises a sequence that is substantially complementary to a region of the target C3 RNA. The two strands forming the duplex structure can be derived from a single RNA molecule having at least one self-complementary region, or can be formed by two or more separate RNA molecules. In the case where the double-stranded region is formed by a single molecule, the molecule can have a duplex structure (referred to herein as a "hairpin loop") formed by one strand of a single-stranded nucleotide chain at the 3'-end and the other strand at the corresponding 5'-end. In some embodiments of the present invention, the hairpin configuration comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more unpaired nucleotides.When the two substantially complementary strands of a C3 dsRNA agent are composed of separate RNA molecules, these molecules need not be covalently linked, but can be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the connecting structure is referred to as a "linker." The term "siRNA" is also used herein to refer to dsRNA agents as described herein.

[0103] In some embodiments of the invention, a C3 dsRNA agent may comprise a sense and antisense sequence having unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. An end without unpaired nucleotides is referred to as a "blunt end" and has no nucleotide overhangs. If both ends of a dsRNA agent are blunt-ended, the dsRNA is referred to as "blunt-ended." In some embodiments of the invention, the first end of the dsRNA agent is blunt-ended, in some embodiments, the second end of the dsRNA agent is blunt-ended, and in certain embodiments of the invention, both ends of the C3 dsRNA agent are blunt-ended.

[0104] In some embodiments of the dsRNA reagent of the present invention, the dsRNA does not have one or two blunt ends. In this case, there is at least one unpaired nucleotide at the end of one chain of the dsRNA reagent. For example, when the 3'-end of one chain of the dsRNA extends beyond the 5'-end of the other chain, there is a nucleotide overhang, and vice versa. The dsRNA may comprise an overhang of at least 1, 2, 3, 4, 5, 6 or more nucleotides. The nucleotide overhang may comprise or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. It should be understood that in some embodiments, the nucleotide overhang is on the sense strand of the dsRNA reagent, on the antisense strand of the dsRNA reagent, or at both ends of the dsRNA reagent, and the nucleotides of the overhang may be present at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the dsRNA. In certain embodiments of the present invention, one or more nucleotides in the overhang are replaced by nucleoside phosphorothioates.

[0105] As used herein, the term "antisense strand" or "guide strand" refers to the strand of a C3 dsRNA agent that comprises a region that is substantially complementary to a C3 target sequence. As used herein, the term "sense strand" or "passenger strand" refers to the strand of a C3 dsRNA agent that comprises a region that is substantially complementary to a region of the antisense strand of a C3 dsRNA agent.

[0106] Modification

[0107] In some embodiments of the present invention, the RNA of the C3 RNAi agent is chemically modified to obtain enhanced stability and / or one or more other beneficial properties. The nucleic acids in certain embodiments of the present invention can be synthesized and / or modified by methods known in the art, for example, see "Current protocols in Nucleic Acid Chemistry," Beaucage, SL et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of the C3 dsRNA agents of the present invention include, for example: (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, reverse ligation, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.); (b) base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base-pair with an expanded library of partners, deletion of bases (abasic nucleotides), or conjugated bases; (c) sugar modifications (e.g., at the 2' or 4' position) or replacement of sugars; and (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNA compounds useful in certain embodiments of the C3 dsRNA agents, C3 antisense polynucleotides, and C3 sense polynucleotides of the present invention include, but are not limited to, RNAs comprising modified backbones or lacking natural internucleoside linkages. As a non-limiting example, RNAs with backbone modifications may not have a phosphorus atom in their backbone. RNAs without a phosphorus atom in their internucleoside backbones may be referred to as oligonucleosides. In certain embodiments of the present invention, modified RNAs have a phosphorus atom in their internucleoside backbones.

[0108] It should be understood that the term "RNA molecule" or "RNA" or "ribonucleic acid molecule" includes not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA, which include one or more ribonucleotides / ribonucleoside analogs or derivatives as described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules can be modified in the core base structure or the ribose-phosphate backbone structure (e.g., as described below), and molecules containing ribonucleoside analogs or derivatives must retain the ability to form duplexes. As non-limiting examples, the RNA molecule may also include at least one modified ribonucleoside, including but not limited to 2'-O-methyl modified nucleosides, nucleosides comprising 5' thiophosphate groups, terminal nucleosides connected to cholesterol derivatives or dodecanoic acid bisdecylamide groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino modified nucleosides, 2'-alkyl modified nucleosides, morpholino nucleosides, phosphoramidates or non-natural bases comprising nucleosides, or any combination thereof. In some embodiments of the present invention, the RNA molecule includes the following number of modified ribonucleosides: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to the total length of the ribonucleoside of the C3 dsRNA agent molecule. For each of the multiple modified ribonucleosides in such an RNA molecule, the modifications need not be identical.

[0109] In some embodiments, the dsRNA reagents, C3 antisense polynucleotides and / or C3 sense polynucleotides of the present invention can include one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester bonds. The term "independent selection" as used herein refers to selected elements, such as modified nucleotides, non-phosphodiester bonds, etc., and refers to two or more selected elements that can be identical to each other but do not have to be identical to each other. As used herein, "nucleotide bases", "nucleotides" or "core bases" are heterocyclic pyrimidine or purine compounds, which are standard components of all nucleic acids and include bases that form nucleotides: adenine (a), guanine (g), cytosine (c), thymine (t) and uracil (u). Core bases can be further modified to include (but are not intended to be limiting): universal bases, hydrophobic bases, mixed bases, bases with expanded size and fluorinated bases. The terms "ribonucleotides" or "nucleotides" can be used herein to refer to unmodified nucleotides, modified nucleotides or alternative moieties. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil may be replaced by other moieties without significantly altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such a replacement moiety.

[0110] In one embodiment, the modified RNA contemplated for use in the methods and compositions described herein is a peptide nucleic acid (PNA), which has the ability to form a desired duplex structure and allow or mediate specific degradation of the target RNA via the RISC pathway. In certain embodiments of the invention, the C3 RNA interfering agent comprises a single-stranded RNA that interacts with the target C3 RNA sequence to guide the cleavage of the target C3 RNA.

[0111] The RNA backbone of modification can include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidate and aminoalkylphosphoramidate), thiophosphoramidate, thioalkylphosphonates, thioalkylphosphotriester, and boric acid phosphate (it has normal 3'-5' connection, and these 2'-5' connection analogs, and those with inverted polarity, wherein adjacent nucleoside units are connected with 3'-5' to 5'-3' or 2'-5' to 5'-2' form). Various salts, mixed salts and free acid forms are also included. The method for preparing phosphorus-containing bonds is the conventional implementation means of this area, and such method can be used to prepare the C3 dsRNA reagent of some modifications of the present invention, the C3 antisense polynucleotides of some modifications and / or the C3 justice polynucleotides of some modifications.

[0112] The modified RNA backbone that does not contain a phosphorus atom has a backbone formed by a short-chain alkyl or cycloalkyl nucleoside linkage, a mixed heteroatom and an alkyl or cycloalkyl nucleoside linkage, or one or more short-chain heteroatom or heterocyclic nucleoside linkages. It includes those with morpholine bonds (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; methyl and thiomethyl backbones; methylenemethyl and thiomethyl backbones; backbones containing olefins; sulfamate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other portions mixed with N, O, S and CH2 components. Methods for preparing modified RNA backbones that do not contain phosphorus atoms are conventional practices in the art, and such methods can be used to prepare certain modified C3 dsRNA agents of the present invention, certain modified C3 antisense polynucleotides and / or certain modified C3 sense polynucleotides.

[0113] In certain embodiments of the present invention, RNA mimics are included in C3 dsRNA, C3 antisense polynucleotides and / or C3 sense polynucleotides, such as, but not limited to, replacing the sugar and internucleoside linkages (i.e., backbones) of nucleotide units with new groups. In such embodiments, the base units are retained to hybridize with suitable C3 nucleic acid target compounds. One such oligomeric compound (an RNA mimic that has been shown to have excellent hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The core base is retained and is bound directly or indirectly to the nitrogen atoms of the backbone amide portion. Methods for preparing RNA mimics are conventional practices in the art, and such methods can be used to prepare certain modified C3 dsRNA agents of the present invention.

[0114] Some embodiments of the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [referred to as a methylene(methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2- [where the natural phosphodiester backbone is represented as -OPO-CH2-]. Methods for preparing RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare certain modified C3 dsRNA agents, certain C3 antisense polynucleotides, and / or certain C3 sense polynucleotides of the invention.

[0115] The modified RNA may also comprise one or more substituted sugar moieties. The C3 dsRNA, C3 antisense polynucleotide, and / or C3 sense polynucleotide of the present invention may comprise one of the following at the 2' position: OH; F; O-, -S-, or N-alkyl; O-, -S-, or N-alkenyl; O-, -S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include: O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) nCH3)]2, wherein n and m are 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino; substituted silyl, RNA cleavage group, reporter group, intercalator; a group for improving the pharmacokinetic properties of the C3 dsRNA agent; or a group for improving the pharmacodynamic properties of the C3 dsRNA agent, C3 antisense polynucleotide and / or C3 sense polynucleotide, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminoethoxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE). Methods for preparing modified RNAs such as those described are routinely practiced in the art, and such methods can be used to prepare certain modified C3 dsRNA agents of the invention.

[0116] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the C3 dsRNA agents, C3 antisense polynucleotides of the present invention, other positions of the C3 sense polynucleotides and / or C3 sense polynucleotides, particularly the 3' position of the sugar on the 3' terminal nucleotide or in the 2'-5' linked C3 dsRNA, C3 antisense polynucleotides or C3 sense polynucleotides, and the 5' position of the 5' terminal nucleotide. The C3 dsRNA agents, C3 antisense polynucleotides and / or C3 sense polynucleotides can also have sugar mimics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Methods for preparing modified RNAs such as those described are routinely practiced in the art, and such methods can be used to prepare certain modified C3 dsRNA agents, C3 antisense polynucleotides and / or C3 sense polynucleotides of the present invention.

[0117] In some embodiments, C3 dsRNA agents, C3 antisense polynucleotides, and / or C3 sense polynucleotides can include nucleobase (often referred to in the art as simply "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine. Adenine, 5-uracil (pseudouracil), 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines; 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-azaguanine and 7-azaadenine and 3-azaguanine and 3-azaadenine. Additional nucleobases that can be included in certain embodiments of the C3 dsRNA agents of the invention are known in the art, see, for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, Ed. John Wiley & Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Methods for preparing dsRNA, C3 antisense strand polynucleotides, and / or C3 sense strand polynucleotides comprising nucleobase modifications and / or substitutions, such as those described herein, are routinely practiced in the art, and such methods can be used to prepare certain modified C3 dsRNA agents, C3 sense polynucleotides, and / or C3 antisense polynucleotides of the invention.

[0118] Certain embodiments of the C3 dsRNA agents, C3 antisense polynucleotides, and / or C3 sense polynucleotides of the present invention include RNA modified to include one or more locked nucleic acids (LNAs). Locked nucleic acids are nucleotides with modified ribose moieties that contain an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to the C3 dsRNA agents, C3 antisense polynucleotides, and / or C3 sense polynucleotides of the present invention can increase stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Methods for preparing dsRNA agents comprising locked nucleic acids, C3 antisense polynucleotides, and / or C3 sense polynucleotides are routinely practiced in the art, and such methods can be used to prepare certain modified C3 dsRNA agents of the present invention. Certain embodiments of the C3 dsRNA compounds, sense polynucleotides, and / or antisense polynucleotides of the present invention include at least one modified nucleotide, wherein the at least one modified nucleotide comprises: a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-seco nucleotide mimetic, a locked nucleotide, a 2'-F-arabino nucleotide, a 2'-methoxyethyl nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, and a 3'-Ome nucleotide, a nucleotide comprising a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base comprising a nucleotide. In some embodiments, the C3 dsRNA compound comprises an E-vinylphosphonate nucleotide at the 5' end of the antisense strand (also referred to herein as the guide strand).

[0119] In certain embodiments of the present invention, at least one modified nucleotide is included at the 3' and 5' ends of the C3 dsRNA compound, the sense polynucleotide, and / or the 3' end of the antisense polynucleotide, wherein the at least one modified nucleotide comprises: an abasic nucleotide, a ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, or an inverted 2'-deoxynucleotide. It is known to those skilled in the art that the inclusion of an abasic or inverted abasic nucleotide at the end of an oligonucleotide can enhance stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 2003; 31(11): 2705-2716. doi: 10.1093 / nar / gkg393).

[0120] In certain embodiments of the present invention, the C3 dsRNA compound or antisense polynucleotide comprises at least one modified nucleotide, wherein the at least one modified nucleotide comprises an open-ring nucleic acid nucleotide (UNA) or / and a glycol nucleic acid nucleotide (GNA). It is known to those skilled in the art that UNA and GNA are thermally unstable chemical modifications that can significantly improve the off-target spectrum of siRNA compounds (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018; 9(1): 723. doi: 10.1038 / s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010; 6: 862–70).

[0121] In certain embodiments, the present invention relates to open-ring nucleic acid (UNA) oligomers for therapeutic use. Open-ring nucleic acid (UNA) is an acyclic analog of RNA in which the bond between the C2' and C3' atoms of the ribose ring has been severed. It has been demonstrated that the incorporation of UNA is well tolerated for siRNA gene silencing activity and can even enhance its activity in some cases (Meghan A. et al. "Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals". Chem. Soc. Rev., 2011, 40, 5680–5689).

[0122] UNA is a heat-labile modification, and replacing ribonucleotides with UNA reduces base pairing strength and duplex stability. Strategic placement of UNA in the seed region of the siRNA antisense strand can reduce off-target activity in gene silencing mechanisms mediated by microRNA (miRNA). miRNAs primarily recognize target genes for gene suppression through base pairing between the antisense seed region (positions 2-8 from the 5' end) and the target mRNA. Each miRNA has the potential to regulate a large number of genes. The siRNA antisense strand loaded by the RNA-induced silencing complex (RISC) can also potentially regulate a large number of unintended genes through miRNA-mediated mechanisms. Therefore, the addition of thermally unstable nucleotides, such as UNA, to the seed region of siRNA can reduce off-target activity (Lam JK, Chow MY, Zhang Y, Leung SW. siRNA Versus miRNA as Therapeutics for Gene Silencing. Mol Ther Nucleic Acids. 2015 Sep 15; 4(9): e252. doi: 10.1038 / mtna.2015.23. PMID: 26372022; PMCID: PMC4877448.). Specifically, such RNA oligonucleotides or RNA oligonucleotide complexes contain at least one UNA nucleobase monomer in the seed region (Narendra Vaish et al. "Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analog". Nucleic Acids Research, 2011, Vol. 39, No. 5 1823–1832).

[0123] According to the present invention, potential advantages of incorporating UNA into RNA oligonucleotides or RNA oligonucleotide complexes include but are not limited to:

[0124] 1. Reduce off-target activity. Adding UNA to the siRNA seed region reduces the base pairing strength of the seed region, thereby reducing potential off-target activity caused by the micro-RNA mechanism.

[0125] 2. UNA is well tolerated in terms of siRNA activity. In some cases, UNA can lead to enhanced activity.

[0126] Exemplary UNA monomers that can be used in this technology include, but are not limited to:

[0127] Invab is an inverted abasic (deoxyribose) residue. For example, in some embodiments, Invab: In some embodiments, the sense strand or antisense strand may include a "terminal cap," which, as used herein, is a non-nucleotide compound or other moiety that can be incorporated at one or more ends of the strand of the RNAi agent disclosed herein, and in some cases can provide certain advantageous properties for the RNAi agent, such as, for example, protection against exonuclease degradation. In some embodiments, an inverted abasic residue (Invab) is added as an end cap (see, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31 (11), 2705-16). End caps are generally known in the art, and in some embodiments, the end cap is present at the 5' end, the 3' end, or both the 5' and 3' ends of the sense strand.

[0128] Another modification can be included in the RNA of the C3 dsRNA agent, C3 antisense polynucleotide and / or C3 sense polynucleotide of certain embodiments of the present invention, which includes one or more ligands, moieties or conjugates chemically linked to the RNA that enhance one or more characteristics of the C3 dsRNA agent, C3 antisense polynucleotide and / or C3 sense polynucleotide, respectively. Non-limiting examples of characteristics that can be enhanced are: C3 dsRNA agent, C3 antisense polynucleotide and / or C3 sense polynucleotide activity, cellular distribution, delivery of C3 dsRNA agent, pharmacokinetic properties of C3 dsRNA agent and cellular uptake of C3 dsRNA agent. In some embodiments of the present invention, the C3 dsRNA agent comprises one or more targeting groups or linking groups, which, in certain embodiments of the C3 dsRNA agent of the present invention, are conjugated to the sense strand. A non-limiting example of a targeting group is a compound comprising N-acetyl-galactosamine (GalNAc). The terms "targeting group," "targeting agent," "linking agent," "targeting compound," and "targeting ligand" are used interchangeably herein. In certain embodiments of the present invention, the C3 dsRNA agent comprises a targeting compound conjugated to the 5'-end of the sense strand. In certain embodiments of the present invention, the C3 dsRNA agent comprises a targeting compound conjugated to the 3'-end of the sense strand. In certain embodiments of the present invention, the C3 dsRNA agent comprises a targeting group containing GalNAc. In certain embodiments of the present invention, the C3 dsRNA agent does not comprise a targeting compound conjugated to one or both of the 3'-end and the 5'-end of the sense strand. In certain embodiments of the present invention, the C3 dsRNA agent does not comprise a targeting compound conjugated to one or both of the 5'-end and the 3'-end of the sense strand.

[0129] Additional targeting and linking agents are well known in the art. For example, targeting and linking agents that can be used in certain embodiments of the present invention include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20: 533-538), fatty chains, such as dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259: 327-330; Svinarchuk et al., Biochimie, 1993, 75: 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0130] Certain embodiments of compositions comprising C3 dsRNA agents, C3 antisense polynucleotides, and / or C3 sense polynucleotides may include ligands that alter the distribution, targeting, and other properties of the C3 dsRNA agents. In some embodiments of compositions comprising C3 dsRNA agents of the invention, the ligand increases affinity for a selected target (e.g., a molecule, cell or cell type, compartment, such as a cell or organ compartment, tissue, organ, or body region), for example, compared to a species in which such a ligand is absent. Ligands useful in the compositions and / or methods of the invention can be naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins), carbohydrates (e.g., glucans, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, such as a synthetic polyamino acid or polyamine. Examples of polyamino acids are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0131] The ligands included in the compositions and / or methods of the present invention may comprise a targeting group, non-limiting examples of which are cell or tissue targeting agents, e.g., lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells or liver cells. The targeting group may be thyrotropin, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamate, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0132] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine); artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecanol, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl or phenoxazine and peptide conjugates (e.g., antennary peptides, Tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino groups, alkyl groups, substituted alkyl groups, radiolabels, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazoles, bisimidazoles, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocyclic Eu 3+ complex), dinitrophenyl, HRP or AP.

[0133] The ligands included in the compositions and / or methods of the present invention can be proteins, such as glycoproteins or peptides, such as molecules with specific affinity for a co-ligand, or antibodies, such as antibodies that bind to specific cell types such as cancer cells, endothelial cells, cardiomyocytes, or osteocytes. In embodiments of the compositions and / or methods of the present invention, useful ligands can be hormones or hormone receptors. In embodiments of the compositions and / or methods of the present invention, useful ligands can be lipids, lectins, carbohydrates, vitamins, coenzymes, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. In embodiments of the compositions and / or methods of the present invention, useful ligands can be substances that increase the uptake of C3 dsRNA agents into cells, for example, by destroying the cytoskeleton of the cells (e.g., by destroying the microtubules, microfilaments, and / or intermediate filaments of the cells). Non-limiting examples of such agents are: paclitaxel, vincristine, vinblastine, cytochalasin, nocodazole, activator of microfilament polymerization, limnidazole A, phalloidin, poliporin A, indanine, and troponin.

[0134] In some embodiments, the ligands attached to the C3 dsRNA agents of the present invention are used as pharmacokinetic (PK) modulators. Examples of PK modulators that can be used in the compositions and methods of the present invention include, but are not limited to, lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, bile acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, serum protein binding aptamers, and the like. Oligonucleotides containing many phosphorothioate bonds are also known to bind to serum proteins, and thus, short oligonucleotides containing multiple phosphorothioate bonds in the backbone, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, can also be used as ligands in the compositions and / or methods of the present invention.

[0135] C3 dsRNA reagent composition

[0136] In some embodiments of the present invention, the C3 dsRNA agent is in a composition. The composition of the present invention may comprise one or more C3 dsRNA agents and optionally one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc. Non-limiting examples of targeting agents useful in some embodiments of the present methods are agents that guide the C3 dsRNA agent of the present invention to and / or into cells to be treated. The choice of targeting agent will depend on the following factors: the nature of the C3-related disease or condition, and the target cell type. In one non-limiting example, in some embodiments of the present invention, it may be necessary to target the C3 dsRNA agent to and / or into hepatocytes. It should be understood that in some embodiments of the present methods, the therapeutic agent comprises a C3 dsRNA agent having only a delivery agent, such as a delivery agent comprising N-acetylgalactosamine (GalNAc), without any additional linking elements. For example, in some aspects of the invention, a C3 dsRNA agent can be linked to a delivery compound comprising GalNAc and contained in a composition comprising a pharmaceutically acceptable carrier and administered to a cell or subject without any detectable label or targeting agent, etc., linked to the C3 dsRNA agent.

[0137] In cases where the C3 dsRNA agents of the present invention are administered together with and / or attached to one or more delivery agents, targeting agents, labeling agents, etc., those skilled in the art will understand and be able to select and use appropriate agents for use in the methods of the present invention. Labeling agents can be used in certain methods of the present invention to determine the location of the C3 dsRNA agent in cells and tissues, and can be used to determine the cell, tissue, or organ location of a therapeutic composition containing a C3 dsRNA agent that has been administered in the methods of the present invention. Means for attaching and using labeling agents such as enzyme labels, dyes, radioactive labels, etc. are well known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, the labeling agent is attached to one or both of the sense polynucleotide and the antisense polynucleotide contained in the C3 dsRNA agent.

[0138] Delivery of C3 dsRNA Agents and C3 Antisense Polynucleotide Agents

[0139] Certain embodiments of the methods of the present invention include delivering a C3 dsRNA agent into a cell. As used herein, the term "delivery" refers to promoting or influencing cellular uptake or absorption. Absorption or uptake of the C3 dsRNA agent can occur through independent diffusion or active cellular processes, or through the use of delivery agents, targeting agents, etc. that can be associated with the C3 dsRNA agent of the present invention. Delivery methods suitable for the methods of the present invention include, but are not limited to, in vivo delivery, wherein the C3 dsRNA agent is injected into a tissue site or administered systemically. In some embodiments of the present invention, the C3 dsRNA agent is attached to a delivery agent.

[0140] Non-limiting examples of methods that can be used to deliver C3 dsRNA agents to cells, tissues, and / or subjects include: C3 dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods, and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents to treat various diseases and conditions, such as, but not limited to, liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, and the like. Detailed information on various delivery methods can be found in publications such as: Nikam, RR & K.R. Gore (2018) Nucleic Acid Ther, 28(4), 209-224 Aug 2018; Springer AD & S.F. Dowdy (2018) Nucleic Acid Ther. Jun 1; 28(3): 109–118; Lee, K. et al., (2018) Arch Pharm Res, 41(9), 867-874; and Nair, JK et al., (2014) J. Am. Chem. Soc. 136: 16958-16961, the contents of which are incorporated herein by reference.

[0141] Some embodiments of the present invention include using lipid nanoparticles (LNPs) to deliver the C3 dsRNA agents of the present invention to cells, tissues, and / or subjects. LNPs are commonly used for in vivo delivery of C3 dsRNA agents, including therapeutic C3 dsRNA agents. One benefit of using LNPs or other delivery agents is that when LNPs or other delivery agents are used to deliver to subjects, the stability of the C3 RNA agent increases. In some embodiments of the present invention, LNPs comprise cationic LNPs loaded with one or more C3 RNAi molecules of the present invention. The LNPs comprising the C3 RNAi molecules are administered to subjects, and the LNPs and the attached C3 RNAi molecules are taken up by cells through endocytosis, and their presence causes the release of RNAi triggering molecules, thereby mediating RNAi.

[0142] Another non-limiting example of a delivery agent that can be used in embodiments of the present invention to deliver the C3 dsRNA agent of the present invention to cells, tissues, and / or objects is a GalNAc-containing agent that is linked to the C3 dsRNA agent of the present invention and delivers the C3 dsRNA agent to cells, tissues, and / or objects. PCT application WO2020191183 A1 discloses examples of other delivery agents that can be used in certain embodiments of the methods and compositions of the present invention. A non-limiting example of a GalNAc targeting ligand that can be used in the compositions and methods of the present invention to deliver the C3 dsRNA agent to cells is a targeting ligand cluster. Examples of targeting ligand clusters proposed herein include a GalNAc ligand (GLO) having a phosphodiester bond and a GalNAc ligand (GLS) having a phosphorothioate bond. The term "GLX-n" may be used herein to represent an example of a linked GalNAC-containing compound, but is not limited to the following compounds: GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16, the structure of each of which is shown below. In the figure below, the connection position of the GalNAc targeting ligand to the RNAi agent of the present invention is on the far right of each targeting ligand. It will be understood that any of the RNAi and dsRNA molecules of the present invention can be linked to GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, and the structures of GLS-1 to GLS-2 and GLS-3 to GLO-4 are shown below.

[0143] " Heterocycloalkyl " represents a non-aromatic partially saturated or fully saturated ring (for example, 3-10 or 3-7 membered heterocycloalkyl) with a specified number of ring atoms, wherein the ring atoms are composed of one or more heteroatoms (for example, 1, 2, 3 or 4 heteroatoms) selected from N, O and S, and the remaining ring atoms are carbon. A 5-membered heterocycloalkyl is a heterocycloalkyl with 5 ring atoms. A 6-membered heterocycloalkyl is a heterocycloalkyl with 6 ring atoms. Heterocycloalkyl can be monocyclic or polycyclic (for example, bicyclic, tricyclic). The example of heterocycloalkyl includes oxirane, aziridine, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholinyl and thiomorpholinyl. In addition, one ring of polycyclic heterocycloalkyl can be aromatic (for example, aryl or heteroaryl), as long as the polycyclic heterocycloalkyl is attached to the parent structure via non-aromatic carbon or nitrogen atom. For example, 1,2,3,4-tetrahydroquinolin-1-yl (wherein the moiety is attached to the parent structure via a non-aromatic nitrogen atom) is considered a heterocycloalkyl, whereas 1,2,3,4-tetrahydroquinolin-8-yl (wherein the moiety is attached to the parent structure via an aromatic carbon atom) is not considered a heterocycloalkyl. 3-6 In some embodiments of the present invention, the C3 dsRNA is a monocyclic ring. Unless otherwise specified, the lower heterocycloalkyl group is generally preferably a fully saturated carbocyclic ring. In some embodiments of the present invention, in vivo delivery can also be achieved through a β-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. C3 RNAi agents can also be introduced into cells in vitro using methods known in the art, such as electroporation and lipofection. In certain embodiments of the methods of the present invention, C3 dsRNA is delivered in the absence of a targeting agent. These RNAs can be delivered as "naked" RNA molecules. As a non-limiting example, the C3 dsRNA of the present invention can be administered to a subject in a pharmaceutical composition comprising an RNAi agent but not a targeting agent (e.g., a GalNAc targeting compound) to treat a C3-related disease or condition in the subject, such as kidney disease.

[0144] It will be understood that in addition to certain delivery modes described herein, other RNAi delivery modes can be used in conjunction with the embodiments of the C3 RNAi agents and treatment methods described herein, such as, but not limited to, those described herein and those used in the art.

[0145] The C3 dsRNA agents of the present invention can be administered to a subject in an amount and manner effective to reduce the level and activity of a C3 polypeptide in a cell and / or subject. In some embodiments of the methods of the present invention, one or more C3 dsRNA agents are administered to a cell and / or subject to treat a disease or condition associated with C3 expression and activity. In some embodiments, the methods of the present invention comprise administering one or more C3 dsRNA agents to a subject in need of such treatment to alleviate a disease or condition associated with C3 expression in the subject. The C3 dsRNA agents or C3 antisense polynucleotide agents of the present invention can be administered to reduce C3 expression and / or activity in one or more cells in vitro, ex vivo, and in vivo.

[0146] In some embodiments of the present invention, the level of C3 polypeptide in a cell is reduced and its activity is thereby reduced by delivering (e.g., introducing) a C3 dsRNA agent or a C3 antisense polynucleotide agent into a cell. Targeting agents and methods can be used to facilitate delivery of a C3 dsRNA agent or a C3 antisense polynucleotide agent to a specific cell type, cell subtype, organ, spatial region, and / or subcellular region within a cell within a subject. A C3 dsRNA agent can be administered alone or in combination with one or more additional C3 dsRNA agents in certain methods of the present invention. In some embodiments, two, three, four, or more independently selected C3 dsRNA agents are administered to a subject. In certain embodiments of the present invention, a C3 dsRNA agent is administered to a subject in combination with one or more additional therapeutic regimens for treating a C3-related disease or condition to treat a C3-related disease or condition. Non-limiting examples of additional therapeutic regimens are administration of one or more C3 antisense polynucleotides of the present invention, administration of a non-C3 dsRNA therapeutic agent, and behavioral modification. The additional therapeutic regimen can be administered at one or more of the following times: before, simultaneously, and after administration of the C3 dsRNA agent of the invention. It should be understood that "simultaneously," as used herein, means within 5 minutes of time zero, within 10 minutes of time zero, within 30 minutes of time zero, within 45 minutes of time zero, and within 60 minutes of time zero, wherein "time zero" is the time at which the C3 dsRNA agent of the invention is administered to the subject. Non-limiting examples of non-C3 dsRNA therapeutic agents are: additional therapeutic agents, such as, for example, anti-complement component C5 antibodies or antigen-binding fragments thereof (e.g., eculizumab), iRNA targeting complement component C5 and / or C3 peptide inhibitors (e.g., comprusinvastatin), thereby treating subjects with diseases that would benefit from reduced C3 expression, or any combination of the foregoing, and therapeutic agents formulated as a combination of agents for the treatment of paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome in adults and children. Non-limiting examples of behavioral modifications are: dietary regimens, counseling, and exercise regimens. These and other therapeutic agents and behavioral changes are known in the art and can be used to treat a C3 disease or condition in a subject and can also be combined with one or more C3 dsRNA agents of the present invention and administered to a subject to treat a C3 disease or condition. The C3 dsRNA agents of the present invention administered to cells or subjects to treat a C3-related disease or condition can act synergistically with one or more other therapeutic agents or active ingredients, thereby increasing the effectiveness of the one or more therapeutic agents or active ingredients and / or increasing the effectiveness of the C3 dsRNA agent in treating a C3-related disease or condition.

[0147] The therapeutic methods of the present invention comprise administering a C3 dsRNA agent, which can be used prior to the onset of a C3-related disease or condition and / or while a C3-related disease or condition is present, including at the early, middle, and late stages of the disease or condition, and at all times before and after any of these stages. The methods of the present invention can also treat a subject who has previously been treated for a C3-related disease or condition with one or more other therapeutic agents and / or therapeutically active ingredients, where the one or more other therapeutic agents and / or therapeutically active ingredients were unsuccessful, minimally successful, and / or no longer successful in treating the subject's C3-related disease or condition.

[0148] Vector-encoded dsRNA

[0149] In certain embodiments of the present invention, a vector can be used to deliver a C3 dsRNA agent to a cell. The C3 dsRNA agent transcription unit can be contained in a DNA or RNA vector. The preparation and use of such vectors encoding transgenes for delivering sequences to cells and / or subjects are well known in the art. Vectors that result in transient expression of the C3 dsRNA can be used in the methods of the present invention, for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more. The length of transient expression can be determined using conventional methods based on factors such as, but not limited to, the specific vector construct and target cell and / or tissue selected. Such transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed so that they are inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0150] One or more single strands of a C3 dsRNA agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to produce, for example, dsRNA, the two separate expression vectors can be co-introduced into cells using, for example, transfection or infection. In certain embodiments, each separate strand of a C3 dsRNA agent of the present invention can be transcribed from a promoter contained on the same expression vector. In certain embodiments of the present invention, the C3 dsRNA agent is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence, such that the C3 dsRNA agent has a stem-loop structure.

[0151] Non-limiting examples of RNA expression vectors are DNA plasmids or viral vectors. Useful expression vectors in embodiments of the present invention may be compatible with eukaryotic cells. Eukaryotic cell expression vectors are routinely used in the art and are available from many commercial sources. Delivery of the C3 dsRNA expression vector may be systemic, for example, by intravenous or intramuscular administration, by administration to target cells removed from a subject and then reintroducing the target cells into the subject, or by any other means that allows for introduction into the desired target cells.

[0152] Viral vector systems that may be included in embodiments of the method include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV 40 vectors; (f) polyoma viral vectors; (g) papilloma viral vectors; (h) picornaviral vectors; (i) poxviral vectors, such as orthopoxviral vectors, such as vaccinia viral vectors or avian poxviral vectors, such as canarypox or fowlpox viral vectors; (j) helper-dependent or gutless adenoviral vectors. Constructs for recombinant expression of C3 dsRNA agents may include regulatory elements, such as promoters, enhancers, and the like, which may be selected to provide constitutive or regulated / inducible expression. Viral vector systems and the use of promoters and enhancers, etc., are conventional in the art and may be used in conjunction with the methods and compositions described herein.

[0153] Certain embodiments of the present invention include the use of viral vectors to deliver C3 dsRNA agents into cells. Many adenovirus-based delivery systems are routinely used in the art for delivery to, for example, the lungs, liver, central nervous system, endothelial cells, and muscle. Non-limiting examples of viral vectors that can be used in the methods of the present invention are: AAV vectors, poxviruses such as vaccinia virus, modified Ankara virus (MVA), NYVAC, avian pox such as fowlpox or canarypox virus.

[0154] Certain embodiments of the present invention include methods for delivering C3 dsRNA agents into cells using vectors, and such vectors can be in pharmaceutically acceptable carriers, which can, but need not, include a sustained-release matrix in which the gene delivery vector is embedded. In some embodiments, the vectors used to deliver C3 dsRNA can be produced by recombinant cells, and the pharmaceutical compositions of the present invention can include one or more cells that produce the C3 dsRNA delivery system.

[0155] Pharmaceutical compositions containing C3 dsRNA or ssRNA agents

[0156] Certain embodiments of the present invention include the use of pharmaceutical compositions containing a C3 dsRNA agent or a C3 antisense polynucleotide agent and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing a C3 dsRNA agent or a C3 antisense polynucleotide agent can be used in the methods of the present invention to reduce C3 gene expression and C3 activity in cells, and can be used to treat C3-related diseases or conditions. Such pharmaceutical compositions can be formulated based on the mode of delivery. Non-limiting examples of formulations for delivery modes are: compositions formulated for subcutaneous delivery, compositions formulated for systemic administration via parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, compositions formulated for direct delivery to the brain, and the like. The pharmaceutical compositions of the present invention can be administered in one or more ways to deliver the C3 dsRNA agent or C3 antisense polynucleotide agent to cells, for example: surface (e.g., via a transdermal patch); pulmonary, such as by inhalation or insufflation of a powder or aerosol, including via a nebulizer; intra-airway, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous, for example, by implantation; or intracranial, for example, by intraparenchymal; intrathecal or intraventricular administration. C3 dsRNA agents or C3 antisense polynucleotide agents can also be delivered directly to target tissues, for example, directly to the liver, directly to the kidneys, etc. It is understood that "delivering a C3 dsRNA agent" or "delivering a C3 antisense polynucleotide agent" to a cell includes delivering a C3 dsRNA agent or C3 antisense polynucleotide agent, directly expressing a C3 dsRNA agent in a cell, and expressing a C3 dsRNA agent from an encoding vector delivered to a cell, or any suitable means by which a C3 dsRNA or C3 antisense polynucleotide agent appears in the cell. The preparation and use of the formulations and the means for delivering inhibitory RNA are well known and commonly used in the art.

[0157] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term explicitly excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are typically magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The agents included in the pharmaceutical formulations are further described below. As used herein, terms such as "pharmacologically effective amount," "therapeutically effective amount," and "effective amount" refer to an amount of a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention that produces the desired pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by at least 10%, then the therapeutically effective amount of the agent for treating that disease or disorder is the amount required to reduce that parameter by at least 10%. For example, a therapeutically effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent can reduce C3 polypeptide levels by at least 10%. Pharmaceutical compositions can include dsRNAi agents such as duplexes AV00375 to AV00447 shown in Table 1. In some embodiments, preferred dsRNAi agents include, for example, duplexes AV00375, AV00413, AV00416, or AV00429. In other embodiments, preferred dsRNAi agents of Table 2 include, for example, AV00375, AV00413, AV00416, or AV00429.

[0158] effective dose

[0159] In some aspects, the methods of the present invention comprise contacting a cell with an effective amount of a C3 dsRNA agent or a C3 antisense polynucleotide agent to reduce C3 gene expression in the contacted cell. Certain embodiments of the methods of the present invention comprise administering a C3 dsRNA agent or a C3 antisense polynucleotide agent to a subject in an amount effective to reduce C3 gene expression and treat a C3-related disease or condition in the subject. As used herein, an "effective amount" is an amount necessary or sufficient to achieve the desired biological effect for reducing C3 expression and / or for treating a C3-related disease or condition. For example, an effective amount of a C3 dsRNA agent or a C3 antisense polynucleotide agent for treating a C3-related disease or condition can be: (i) an amount required to slow or stop the progression of the disease or condition; or (ii) to reverse, reduce, or eliminate one or more symptoms of the disease or condition. In some aspects of the present invention, an effective amount is an amount of a C3 dsRNA agent or a C3 antisense polynucleotide agent that, when administered to a subject in need of treatment for a C3-related disease or condition, results in a therapeutic response for the prevention and / or treatment of the disease or condition. According to some aspects of the invention, an effective amount is an amount of a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention that, when combined or co-administered with another therapeutic treatment for a C3-related disease or condition, results in a therapeutic response that prevents and / or treats the disease or condition. In some embodiments of the invention, the biological effect of treating a subject with a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention can be an improvement and / or complete elimination of symptoms caused by a C3-related disease or condition. In some embodiments of the invention, the biological effect is complete elimination of a C3-related disease or condition, as demonstrated, for example, by a diagnostic test indicating that the subject does not have a C3-related disease or condition. Non-limiting examples of detectable physiological symptoms include a decrease in lipid accumulation in the liver of a subject following administration of an agent of the invention. Other art-known methods for assessing the status of a C3-related disease or condition can be used to determine the effect of the agents and / or methods of the invention on a C3-related disease or condition.

[0160] The effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent that reduces C3 polypeptide activity to a level that treats a C3-related disease or condition is typically determined in clinical trials, where effective doses are established for test and control populations in blinded studies. In some embodiments, an effective amount is an amount that results in a desired response, such as a reduction in the amount of a C3-related disease or condition in cells, tissues, and / or subjects suffering from the disease or condition. Thus, an effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent for treating a C3-related disease or condition that can be treated by reducing C3 polypeptide activity can be an amount that, when administered, reduces the amount of C3 polypeptide activity in a subject to a level below that which would be present in the cell, tissue, and / or subject in the absence of administration of the C3 dsRNA agent or C3 antisense polynucleotide agent. In certain aspects of the invention, the level of C3 polypeptide activity and / or C3 gene expression present in cells, tissues, and / or subjects that have not been exposed to or administered a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention is referred to as a "control" amount. In some embodiments of the methods of the present invention, the control amount for a subject is the subject's pre-treatment amount; in other words, the subject's level prior to administration of the C3 agent can be the subject's control level and is used to compare its C3 polypeptide activity and / or C3 gene expression level after administration of the siRNA to the subject. In the case of treating a C3-related disease or condition, the desired response can be a reduction or elimination of one or more symptoms of the disease or condition in cells, tissues, and / or the subject. The reduction or elimination can be temporary or permanent. It should be understood that methods of determining C3 polypeptide activity, C3 gene expression, symptom assessment, clinical testing, and the like can be used to monitor the status of a C3-related disease or condition. In some aspects of the present invention, the desired response to treating a C3-related disease or condition is to delay the onset of the disease or condition or even prevent the onset of the disease or condition.

[0161] The effective amount of a compound that reduces the activity of a C3 polypeptide can also be determined by evaluating the physiological effects of administering a C3 dsRNA agent or a C3 antisense polynucleotide agent on a cell or subject, such as a reduction in a C3-related disease or condition following administration. Assays and / or symptom monitoring of subjects can be used to determine the efficacy of the C3 dsRNA agents or C3 antisense polynucleotide agents of the present invention (which can be administered in the pharmaceutical compounds of the present invention) and to determine whether there is a response to treatment. A non-limiting example is one or more serum lipid profile tests known in the art. Another non-limiting example is that before and after treating a subject with a C3 dsRNA agent of the present invention, one or more renal function tests known in the art can be used to determine the status of a C3-related kidney disease or condition in the subject. In another non-limiting example, one or more tests for acquired hemolytic anemia, hemoglobinuria, bone marrow failure, and thrombophilia (tendency to form blood clots) known in the art are used to determine the status of a C3-related disease in the subject. In this embodiment, the disease comprises paroxysmal nocturnal hemoglobinuria (PNH), and the test is used to determine the level of hemolysis in the subject before and after treatment of the subject with a C3 dsRNA agent of the invention.

[0162] Some embodiments of the present invention include methods for determining the efficacy of a C3 dsRNA agent or C3 antisense polynucleotide agent administered to a subject to treat a C3-related disease or condition by assessing and / or monitoring one or more "physiological characteristics" of a C3-related disease or condition in the subject. A non-limiting example of a physiological characteristic of a C3-related disease or condition is that many patients also experience an increased risk of renal failure and thromboembolism. Standard methods for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, and the like. The syndrome is defined by the presence of hemolysis, elevated liver enzymes, and low platelet counts.

[0163] It will be appreciated that the amount of a C3 dsRNA agent or C3 antisense polynucleotide agent administered to a subject can be modified based, at least in part, on the results of such determinations of the subject's disease and / or condition state and / or physiological characteristics. The therapeutic amount can be varied, for example, by increasing or decreasing the amount of the C3-dsRNA agent or C3 antisense polynucleotide agent by changing the composition of the C3 dsRNA agent or C3 antisense polynucleotide agent, by changing the route of administration, by changing the time of administration, and the like. The effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent will vary depending on the specific condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of co-treatments (if any), the specific route of administration, and other factors within the knowledge and expertise of a healthcare practitioner. For example, the effective amount can depend on the desired level of C3 polypeptide activity and / or C3 gene expression to be effective in treating a C3-related disease or condition. A skilled artisan can empirically determine the effective amount of a particular C3 dsRNA agent or C3 antisense polynucleotide agent for use in the methods of the present invention without undue experimentation. In combination with the teachings provided herein, by selecting from a variety of C3 dsRNA agents or C3 antisense polynucleotide agents of the present invention and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred route of administration, an effective prophylactic or therapeutic treatment regimen can be formulated to effectively treat a particular subject. As used in the embodiments of the present invention, an effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent of the present invention can be an amount that produces a desired biological effect in a cell when contacted with the cell.

[0164] It should be understood that C3 gene silencing can be performed constitutively or by genomic engineering in any cell expressing C3 and determined by any suitable assay. In some embodiments of the invention, C3 gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% by administering a C3 dsRNA agent of the invention. In some embodiments of the invention, C3 gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100% by administering a C3 dsRNA agent of the invention.

[0165] Drug administration

[0166] C3 dsRNA agents and C3 antisense polynucleotide agents are delivered in pharmaceutical compositions at a dose sufficient to inhibit C3 gene expression. In certain embodiments of the present invention, the dose of C3 dsRNA agents or C3 antisense polynucleotide agents is 0.01 to 200.0 mg per kilogram of recipient body weight per day, generally 1 to 50 mg / kg body weight, 5 to 40 mg / kg body weight, 10 to 30 mg / kg body weight, 1 to 20 mg / kg body weight, 1 to 10 mg / kg body weight, 4 to 15 mg / kg body weight, including end values. For example, C3 Each single administration of a dsRNA agent or a C3 antisense polynucleotide agent can be at a dosage of from about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2 mg / kg, 2.1 mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4. 4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5m g / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg , 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg to 50mg / kg body weight.

[0167] A variety of factors can be considered when determining the dosage and timing of delivery of the C3 dsRNA agents of the present invention. The absolute amount of the C3 dsRNA agent or C3 antisense polynucleotide agent delivered will depend on a variety of factors, including co-treatment, number of doses, and individual subject parameters, including age, physical condition, size, and weight. These factors are well known to those of ordinary skill in the art and can be addressed through routine experimentation. In some embodiments, a maximum dose, i.e., the highest safe dose based on sound medical judgment, can be used.

[0168] In some embodiments, the methods of the present invention may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a C3 dsRNA agent or a C3 antisense polynucleotide agent to a subject. In some cases, a dose of a pharmaceutical compound (e.g., comprising a C3 dsRNA agent or comprising a C3 antisense polynucleotide agent) may be administered to a subject at least daily, every other day, weekly, every other week, monthly, etc., and may be administered once a day or more than once a day, for example, 2, 3, 4, 5 or more times within a 24-hour period. The pharmaceutical composition of the present invention may be administered once a day; or the C3 dsRNA agent or C3 antisense polynucleotide agent may be administered as two, three or more sub-doses at appropriate intervals throughout the day, or even using continuous infusion or delivered by a controlled release formulation. In some embodiments of the methods of the present invention, the pharmaceutical composition of the present invention is administered to a subject once a day or more, once a week or more, once a month or more, or once a year or more.

[0169] In certain aspects, the methods of the present invention comprise administering a pharmaceutical compound alone; in combination with one or more other C3 dsRNA agents or C3 antisense polynucleotide agents; and / or in combination with other drug therapies or therapeutic activities or regimens administered to a subject suffering from a C3-related disease or condition. The pharmaceutical compound can be administered in the form of a pharmaceutical composition. The pharmaceutical composition used in the methods of the present invention can be sterile and contain an amount of a C3 dsRNA agent or C3 antisense polynucleotide agent that will reduce the activity of the C3 polypeptide to a level sufficient to produce the desired response in a unit of weight or volume suitable for administration to the subject. The dose of the pharmaceutical composition comprising a C3 dsRNA agent or C3 antisense polynucleotide agent administered to the subject to reduce C3 protein activity can be selected based on various parameters, particularly the mode of administration used and the condition of the subject. Other factors include the desired duration of treatment. If the subject's response to the initial dose is insufficient, a higher dose can be used (or the dose can be effectively increased by a different, more localized delivery route) as tolerated by the patient.

[0170] treat

[0171] As used herein, the terms "prevent" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced C3 gene expression, means that a subject is less likely to develop symptoms associated with such disease, disorder, or condition, such as symptoms associated with a disease or condition caused by or associated with C3 activation, such as C3 glomerulopathy (C3G). The likelihood of developing C3 glomerulopathy (C3G) is reduced, for example, when an individual with one or more C3 glomerulopathy (C3G) risk factors fails to develop the disease, disorder, or condition, or develops symptoms associated with the disease, disorder, or condition to a lesser extent (e.g., by at least about 10% on a scale that would indicate clinically developing the disease or condition), or delays the manifestation of symptoms (e.g., by days, weeks, months, or years) relative to a population of individuals with the same risk factors who do not receive the treatment described herein.

[0172] For C3-associated diseases and disorders where reduction of the level and / or activity of a C3 polypeptide is effective in treating the disease or disorder, the methods and C3 dsRNA agents of the present invention can be used for treatment to inhibit C3 expression. Examples of diseases and conditions that can be treated with the C3 dsRNA agents or C3 antisense polynucleotide agents of the invention and the treatment methods of the invention include, but are not limited to: C3 glomerulopathy (C3G), immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus, lupus nephritis, ischemia / reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), and diseases of other organs known to be associated with complement dysfunction, such as age-related macular degeneration (AMD), rheumatoid arthritis (RA), anti-neutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegenerative diseases. Such diseases and disorders may be referred to herein as "C3-associated diseases and disorders" and "diseases and disorders caused and / or modulated by C3."

[0173] In certain aspects of the invention, a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention can be administered to a subject at one or more times before or after diagnosis of a C3-related disease or condition. In some aspects of the invention, a subject is at risk of having or developing a C3-related disease or condition. A subject at risk for developing a C3-related disease or condition is a subject that has an increased likelihood of developing a C3-related disease or condition compared to a control risk for developing a C3-related disease or condition. In some embodiments of the invention, the level of risk is statistically significant compared to the control level of risk. At-risk subjects can include, for example: subjects who are or will be subjects with a pre-existing disease and / or genetic abnormality that makes the subject more susceptible to a C3-related disease or condition than a control subject without a pre-existing disease or genetic abnormality; subjects with a family and / or personal history of a C3-related disease or condition; and subjects who have previously received treatment for a C3-related disease or condition. It will be understood that a pre-existing disease and / or genetic abnormality that renders a subject more susceptible to a C3-related disease or condition may be a disease or genetic abnormality that, when present, has been previously determined to be associated with a higher likelihood of developing a C3-related disease or condition.

[0174] It should be understood that a C3 dsRNA agent or C3 antisense polynucleotide agent can be administered to a subject based on the medical condition of the individual subject. For example, the healthcare provider providing the subject can assess the C3 level measured in a sample obtained from the subject and determine that it is desirable to reduce the C3 level of the subject by administering the C3 dsRNA agent or C3 antisense polynucleotide agent of the present invention. In a non-limiting example, a biological sample, such as a blood or serum sample, can be obtained from the subject, and the C3 level of the subject can be determined in the sample. A C3 dsRNA agent or C3 antisense polynucleotide agent is administered to the subject, and a blood or serum sample is obtained from the subject after administration, and the C3 level is determined using the sample, and the result is compared with the result determined in the sample before administration (previous). A reduction in the C3 level of the subject in the subsequent sample compared to the pre-administration level indicates the efficacy of the administered C3 dsRNA agent or C3 antisense polynucleotide agent in reducing the C3 level of the subject. In a non-limiting example, hemolysis can be considered a physiological characteristic of a C3-related condition, even if the subject has not been diagnosed with a C3-related disease, such as a disease disclosed herein. A healthcare provider can monitor changes in hemolysis in a subject as a measure of the efficacy of the administered C3 dsRNA agent or C3 antisense polynucleotide agent of the invention. In one non-limiting example, a complement component C3-related disease is paroxysmal nocturnal hemoglobinuria (PNH). PNH may be classic PNH or PNH in the context of another bone marrow failure syndrome and / or myelodysplastic syndrome (MDS), e.g., cytopenia.

[0175] Certain embodiments of the methods of the present invention include adjusting treatment, which includes administering a dsRNA agent or C3 antisense polynucleotide agent of the present invention to a subject based at least in part on an assessment of changes in one or more physiological characteristics of a C3-related disease or condition in the subject caused by the treatment. For example, in some embodiments of the present invention, the effect of a dsRNA agent or C3 antisense polynucleotide agent administered to a subject can be determined and used to help adjust the amount of a dsRNA agent or C3 antisense polynucleotide agent subsequently administered to the subject. In one non-limiting example, a dsRNA agent or C3 antisense polynucleotide agent of the present invention is administered to a subject, and after administration, the subject is measured for thrombosis; and based at least in part on the determined level, it is determined whether a higher amount of the dsRNA agent or C3 antisense polynucleotide agent is needed to enhance the physiological effect of the administered agent, such as to reduce or further reduce hemolysis in the subject. In another non-limiting example, a dsRNA agent or C3 antisense polynucleotide agent of the present invention is administered to a subject, and after administration, the subject's hemolysis is determined, and based at least in part on the determined level, it is expected that a lower amount of the dsRNA agent or C3 antisense polynucleotide agent will be administered to the subject.

[0176] Thus, some embodiments of the present invention include assessing changes in one or more physiological characteristics resulting from a previous treatment of a subject to adjust the amount of the dsRNA agent or C3 antisense polynucleotide agent of the present invention subsequently administered to the subject. Some embodiments of the methods of the present invention include 1, 2, 3, 4, 5, 6, or more measurements of physiological characteristics of a C3-related disease or condition; assessing and / or monitoring the efficacy of the administered C3 dsRNA agent or C3 antisense polynucleotide agent of the present invention; and optionally using the results of the measurements to adjust one or more of the following: the dosage, dosing regimen, and / or dosing frequency of the dsRNA agent or C3 antisense polynucleotide agent of the present invention for treating a C3-related disease or condition in the subject. In some embodiments of the methods of the present invention, the desired result of administering an effective amount of the dsRNA agent or C3 antisense polynucleotide agent of the present invention to the subject is a decrease in the subject's C3 transcript level, plasma C3 level, or C3 gene expression compared to a previous determination for the subject.

[0177] As used herein, the terms "treat," "therapeutic," or "therapeutic" when applied to a C3-related disease or disorder may refer to prophylactic treatment, reducing the likelihood that a subject will develop a C3-related disease or disorder, and may also refer to treatment after a subject has developed a C3-related disease or disorder in order to eliminate or reduce the level of the C3-related disease or disorder, prevent the C3-related disease or disorder from becoming more severe, and / or slow the progression of the C3-related disease or disorder in a subject compared to a subject in the absence of a therapy that reduces the activity of a C3 polypeptide in the subject.

[0178] Certain embodiments of the agents, compositions, and methods of the present invention can be used to inhibit C3 gene expression. As used herein, with respect to expression of the C3 gene, the terms "inhibit," "silence," "reduce," "downregulate," and "knockdown" refer to altering the expression of the C3 gene, for example, by one or more of the following: when a cell, cell population, tissue, organ, or subject is contacted with (e.g., treated with) a C3 dsRNA agent or a C3 antisense polynucleotide agent of the present invention, the level of RNA transcribed from the gene, the level of activity of C3 expressed, and the level of C3 polypeptide, protein, or protein subunit translated from mRNA in the cell, cell population, tissue, organ, or subject is reduced compared to a control level of RNA transcribed from the C3 gene, a control level of activity of C3 expressed, or a control level of C3 translated from mRNA, respectively. In some embodiments, the control level is the level in a cell, tissue, organ, or subject that has not been contacted with (e.g., treated with) a C3 dsRNA agent or a C3 antisense polynucleotide agent.

[0179] Application method

[0180] A variety of routes of administration of C3 dsRNA agents or C3 antisense polynucleotide agents can be used in the methods of the present invention. The choice of a particular mode of delivery will depend, at least in part, on the specific condition being treated and the dosage required for therapeutic efficacy. In general, the methods of the present invention can be implemented using any medically acceptable mode of administration, meaning any mode that produces an effective therapeutic level for a C3-related disease or condition without causing clinically unacceptable side effects. In some embodiments of the present invention, C3 dsRNA agents or C3 antisense polynucleotide agents can be administered orally, enterally, mucosally, subcutaneously, and / or parenterally. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal (e.g., via a nasogastric tube), transdermal, vaginal, rectal, sublingual, and inhalation. The delivery routes of the present invention may include intrathecal, intraventricular, or intracranial. In some embodiments of the present invention, C3 dsRNA agents or C3 antisense polynucleotide agents can be placed in a sustained-release matrix and administered by placing the matrix in a subject. In some aspects of the present invention, a C3 dsRNA agent or a C3 antisense polynucleotide agent can be delivered to a subject's cells using nanoparticles coated with a delivery agent that targets specific cells or organelles. Various delivery methods, methods, and agents are known in the art. Non-limiting examples of delivery methods and delivery agents are provided elsewhere herein. In some aspects of the present invention, the term "delivery" with respect to a C3 dsRNA agent or a C3 antisense polynucleotide agent can refer to administering one or more "naked" C3 dsRNA agent or C3 antisense polynucleotide agent sequences to a cell or subject. In certain aspects of the present invention, "delivery" refers to administering to a cell or subject by transfection, delivering a cell containing a C3 dsRNA agent or a C3 antisense polynucleotide agent to a subject, delivering a vector encoding a C3 dsRNA agent or a C3 antisense polynucleotide agent to a cell and / or subject, and the like. Delivering a C3 dsRNA agent or a C3 antisense polynucleotide agent using transfection can include administering a vector to a cell and / or subject.

[0181] In some methods of the present invention, one or more C3 dsRNA agents or C3 antisense polynucleotide agents can be administered in the form of a formulation or in a pharmaceutically acceptable solution, which can generally contain pharmaceutically acceptable concentrations of salt, a buffer, a preservative, a compatible carrier, an adjuvant, and optionally other therapeutic ingredients. In some embodiments of the present invention, the C3 dsRNA agent or C3 antisense polynucleotide agent can be formulated for simultaneous administration with another therapeutic agent. According to the methods of the present invention, the C3 dsRNA agent or C3 antisense polynucleotide agent can be administered in the form of a pharmaceutical composition. Typically, the pharmaceutical composition comprises the C3 dsRNA agent or C3 antisense polynucleotide agent and an optional pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. As used herein, a pharmaceutically acceptable carrier refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (e.g., the ability of the C3 dsRNA agent or C3 antisense polynucleotide agent to inhibit C3 gene expression in a cell or subject). A variety of methods of administering and delivering C3 dsRNA agents or C3 antisense polynucleotide agents for therapeutic use are known in the art and can be used in the methods of the present invention.

[0182] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, while other carriers are known to those skilled in the art. Such preparations may typically contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. In addition, pharmaceutically acceptable salts can be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0183] Some embodiments of the methods of the present invention include administering one or more C3 dsRNA agents or C3 antisense polynucleotide agents directly to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which a C3-related disease or condition is present or may occur, a non-limiting example of which is the liver or kidney. Direct tissue administration can be achieved by direct injection or other means. Many orally delivered compounds naturally enter and pass through the liver and kidneys, and some embodiments of the treatment methods of the present invention include orally administering one or more C3 dsRNA agents to a subject. The C3 dsRNA agent or C3 antisense polynucleotide agent, alone or in combination with other therapeutic agents, can be administered once, or they can be administered multiple times. If administered multiple times, the C3 dsRNA agent or C3 antisense polynucleotide agent can be administered by different routes. For example, although not intended to be limiting, the first (or first few) administrations can be performed subcutaneously, and one or more additional administrations can be oral and / or systemic.

[0184] For embodiments of the present invention in which systemic administration of a C3 dsRNA agent or C3 antisense polynucleotide agent is desired, the C3 dsRNA agent or C3 antisense polynucleotide agent can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations can be presented in unit dosage form, such as ampoules or multi-dose containers, with or without added preservatives. C3 dsRNA agent formulations (also referred to as pharmaceutical compositions) can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain preparatants, such as suspending agents, stabilizers, and / or dispersants.

[0185] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose solution, glucose and sodium chloride solution, lactated Ringer's solution or fixed oils. Intravenous excipients include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose solution), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, etc., may also be present. Other forms of administration, such as intravenous administration, will result in lower dosages. If the subject's response under the initial dose is insufficient, a higher dosage (or effectively increasing the dosage by different, more localized delivery routes) can be adopted within the scope allowed by patient tolerance. Multiple doses per day can be used as needed to achieve appropriate systemic or local levels of one or more C3 dsRNA agents or C3 antisense polynucleotide agents and to achieve appropriate reduction in C3 protein activity.

[0186] In other embodiments, the methods of the present invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, such as a subject. Exemplary biodegradable implants that can be used according to this method are described in PCT Publication WO 95 / 24929 (incorporated herein by reference), which describes a biocompatible, biodegradable polymer matrix for containing biomacromolecules.

[0187] Both non-biodegradable and biodegradable polymer matrices can be used in the methods of the present invention to deliver one or more C3 dsRNA agents or C3 antisense polynucleotide agents to a subject. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the time period over which release is desired, typically on the order of a few hours to a year or longer. Typically, release over a period of time between a few hours and three to twelve months can be used. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% of its weight in water and is also optionally cross-linked with multivalent ions or other polymers.

[0188] Typically, C3 dsRNA reagents or C3 antisense polynucleotide reagents can be delivered using biodegradable implants by diffusion or by degradation of a polymer matrix in some embodiments of the present invention. Exemplary synthetic polymers for such purposes are well known in the art. Using methods known in the art, biodegradable polymers and non-biodegradable polymers can be used to deliver C3 dsRNA reagents or C3 antisense polynucleotide reagents. Bioadhesive polymers such as bioerodible hydrogels (HS Sawhney, CP Pathak and JA Hubell in Macromolecules, 1993, 26, 581-587) can also be used to deliver C3 dsRNA reagents or C3 antisense polynucleotide reagents to treat C3 related diseases or conditions. Other suitable delivery systems can include timed release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of C3 dsRNA reagents or C3 antisense polynucleotide agents, thereby improving the convenience of the subject and healthcare professionals. Many types of release delivery systems are available and known to those of ordinary skill in the art. See, for example, U.S. Patent Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686. Additionally, pump-based hardware delivery systems may be used, some of which are also suitable for implantation.

[0189] The use of long-term sustained-release implants can be suitable for the prophylactic treatment of subjects and subjects at risk of developing recurrent C3-related diseases or conditions. As used herein, long-term release refers to implants constructed and arranged to deliver therapeutic levels of C3 dsRNA agents or C3 antisense polynucleotide agents for at least 10 days, 20 days, 30 days, 60 days, 90 days, six months, one year, or longer. Long-term sustained-release implants are well known to those of ordinary skill in the art and include some of the release systems described above.

[0190] Therapeutic formulations of C3 dsRNA agents or C3 antisense polynucleotide agents can be prepared for storage by mixing the molecule or compound having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006)] in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as or polyethylene glycol (PEG).

[0191] Cells, subjects, and controls

[0192] The methods of the present invention can be used in conjunction with cells, tissues, organs and / or subjects. In some aspects of the invention, the subject is a human or a vertebrate mammal, including but not limited to dogs, cats, horses, cows, goats, mice, rats and primates, such as monkeys. Thus, the present invention can be used to treat C3-related diseases or conditions in both human and non-human subjects. In some aspects of the invention, the subject can be a farm animal, a zoo animal, a domesticated animal or a non-domesticated animal, and the methods of the present invention can be used in veterinary prevention and treatment protocols. In some embodiments of the invention, the subject is a human and the methods of the present invention can be used in human prevention and treatment protocols.

[0193] Non-limiting examples of subjects to which the present invention can be applied are subjects diagnosed with, suspected of having, or at risk of having a disease or condition associated with higher than desired expression and / or activity of C3, also referred to as "elevated C3 expression levels." Non-limiting examples of diseases and conditions associated with higher than desired levels of C3 expression and / or activity are described elsewhere herein. The methods of the present invention can be applied to subjects who, at the time of treatment, have been diagnosed with the disease or condition, are associated with higher than desired expression and / or activity of C3, or are believed to be at risk of having or developing a disease or condition associated with higher than desired expression and / or activity of C3. In some aspects of the invention, the disease or condition associated with higher than desired levels of C3 expression and / or activity is an acute disease or condition; in certain aspects of the invention, the disease or condition associated with higher than desired levels of C3 expression and / or activity is a chronic disease or condition.

[0194] In one non-limiting example, the C3 dsRNA agent of the present invention is administered to a subject diagnosed with a kidney disease, including but not limited to: C3 glomerulopathy (C3G), immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), ischemia / reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH). The method of the present invention can be applied to a subject who has been diagnosed with the disease or condition at the time of treatment, or a subject who is considered to be at risk of developing or developing the disease or condition.

[0195] In another non-limiting example, the C3 dsRNA agent of the present invention is administered to treat a disease or disorder caused by or associated with C3 activation, or a disease or disorder whose symptoms or progression respond to C3 inactivation. The term "C3-related disease" includes diseases, disorders, or conditions that benefit from reduced C3 expression. Such diseases are generally associated with the physiological characteristics of hemolysis, elevated liver enzymes, and low platelet counts. Kidney diseases include, but are not limited to, C3 glomerulopathy (C3G), immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), ischemia / reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH). In certain embodiments, C3-related diseases include paroxysmal nocturnal hemoglobinuria (PNH).

[0196] In another non-limiting example, a C3 dsRNA agent of the invention is administered to treat a molecule for inhibiting C3 gene expression in a neural cell, such as a neural cell in a subject, such as a mammal, such as a human suffering from a C3-associated neurodegenerative disease, e.g., Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), schizophrenia, Parkinson's disease (PD), and prion diseases, such as Creutzfeldt-Jakob disease (CJD).

[0197] The cell that can be applied the method of the present invention includes in vitro, in vivo, in vitro cell.Cell can be in object, in culture and / or in suspension, or in any other suitable state or condition.The cell that can be applied the method of the present invention can be: liver cell (liver cell), liver cell (hepatocyte), heart cell, pancreatic cell, cardiovascular cell, kidney cell or other types of vertebrate cells, including human and non-human mammalian cells.In some aspects of the present invention, the cell that can be applied the method of the present invention is healthy normal cell, which is not known to be disease cell.In some embodiments of the present invention, the method and composition of the present invention are applied to the cell of liver cell, liver cell, heart cell, pancreatic cell, cardiovascular cell and / or kidney cell.In some aspects of the present invention, control cell is normal cell, but it should be understood that the cell with disease or illness can also be used as control cell in specific cases, for example, when comparing the treated cell with disease or illness and the result of untreated cell with disease or illness etc.

[0198] According to the methods of the present invention, the level of C3 polypeptide activity can be determined and compared to a control level of C3 polypeptide activity. The control can be a predetermined value, which can take a variety of forms. It can be a single cutoff value, such as a median or mean. It can be established based on a comparison group, for example, a group with normal levels of C3 polypeptide and / or C3 polypeptide activity and a group with increased levels of C3 polypeptide and / or C3 polypeptide activity. Another non-limiting example of a comparison group can be a group with one or more symptoms or diagnoses of a C3-related disease or condition versus a group without one or more symptoms or diagnoses of the disease or condition; a group of subjects treated with an siRNA of the present invention versus a group of subjects not treated with an siRNA of the present invention. Typically, the control can be based on apparently healthy normal individuals or apparently healthy cells of an appropriate age group. It should be understood that in addition to predetermined values, the control according to the present invention can be a material sample tested in parallel with the experimental material. Examples include samples from a control population or control samples generated by manufacturing for parallel testing with the experimental samples. In some embodiments of the invention, a control may include cells or subjects that have not been contacted or treated with a C3 dsRNA agent of the invention, in which case the control level of C3 polypeptide and / or C3 polypeptide activity can be compared to the level of C3 polypeptide and / or C3 polypeptide activity in cells or subjects contacted with a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention.

[0199] In some embodiments of the present invention, the control level can be a C3 polypeptide level determined for a subject, wherein C3 polypeptide levels determined for the same subject at different times are compared to the control level. In one non-limiting example, the level of C3 is determined in a biological sample obtained from a subject that has not received C3 treatment according to the present invention. In some embodiments, the biological sample is a serum sample. The C3 polypeptide level determined in the sample obtained from the subject can serve as a baseline or control value for the subject. After administering one or more C3 dsRNA agents to a subject in the treatment methods of the present invention, one or more additional serum samples can be obtained from the subject, and the C3 polypeptide levels in the subsequent one or more samples can be compared to the control / baseline level for the subject. Such comparisons can be used to assess the onset, progression, or regression of a C3-related disease or condition in a subject. For example, a C3 polypeptide level in a baseline sample obtained from a subject that is higher than a level obtained from the same subject after administration of a C3 dsRNA agent or C3 antisense polynucleotide agent according to the present invention indicates regression of the C3-related disease or condition and indicates the efficacy of the administered C3 dsRNA agent according to the present invention in treating the C3-related disease or condition.

[0200] In certain aspects of the present invention, one or more of the values ​​for the level of C3 polypeptide and / or C3 polypeptide activity determined for a subject can serve as a control value and be used to later compare C3 polypeptide and / or C3 activity levels in the same subject, thereby allowing for assessment of changes in "baseline" C3 polypeptide activity in a subject. Thus, the initial C3 polypeptide level and / or initial C3 polypeptide activity level can be used to demonstrate and / or determine the ability of the methods and compounds of the present invention to reduce the level of C3 polypeptide and / or C3 polypeptide activity in a subject, using the initial level as a control level for that subject.

[0201] Using the methods of the present invention, the C3 dsRNA agents and / or C3 antisense polynucleotide agents of the present invention can be administered to a subject. Such dsRNAi agents include, for example, duplexes AV00375 to AV00447 shown in Table 1. In some embodiments, preferred dsRNAi agents include, for example, duplexes AV00375, AV00413, AV00416, or AV00429, and in other embodiments, preferred dsRNAi agents include, for example, AV00375, AV00413, AV00416, or AV00429. The efficacy of the administration and treatment of the present invention can be assessed by reducing the level of C3 polypeptide in a serum sample obtained from the subject after administration and treatment by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, compared to the pre-administration level of C3 polypeptide in a serum sample obtained from the subject at a previous time point, or compared to an untouched control level (e.g., the level of C3 polypeptide in a control serum sample). It will be understood that both the level of C3 polypeptide and the level of C3 polypeptide activity are correlated with the level of C3 gene expression. Certain embodiments of the methods of the present invention comprise administering to a subject a C3 dsRNA and / or C3 antisense agent of the present invention in an amount effective to inhibit C3 gene expression, thereby reducing the level of C3 polypeptide and reducing the level of C3 polypeptide activity in the subject. In some embodiments of the methods of the invention, contacting a cell with an siRNA agent of the invention (also referred to herein as treatment) results in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, %,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,61%,62%,63%,64%,65%,66%,67%,68%,69%,70%,71%,72%,73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%, for example, to below the level of detection in an assay.

[0202] Some embodiments of the present invention include determining the presence, absence, and / or amount (also referred to herein as the level) of a C3 polypeptide in one or more biological samples obtained from one or more subjects. This determination can be used to assess the efficacy of the therapeutic methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of a C3 polypeptide in a biological sample obtained from a subject previously treated with a C3 dsRNA agent and / or a C3 antisense agent of the present invention. A decrease in the level of a C3 polypeptide in a serum sample obtained from a subject after administration and treatment of at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to the pre-administration level of the C3 polypeptide in a serum sample obtained from the subject at a previous time point, or compared to an untouched control level (e.g., the level of the C3 polypeptide in a control serum sample), indicates the efficacy of the treatment administered to the subject.

[0203] In some embodiments of the present invention, the physiological characteristics of a C3-related disease or condition determined for a subject can be used as a control result, and the results of the determination of the physiological characteristics of the same subject at different times can be compared with the control result. In one non-limiting example, the pathological characteristic hemolysis is measured from a subject that has never been given the C3 treatment of the present invention, which can be used as a baseline or control value for the subject. After one or more C3 dsRNA agents are administered to a subject in the treatment methods of the present invention, blood cells are compared with the control / baseline levels of the subject. Such comparisons can be used to assess the onset, progression, or regression of a C3-related disease or condition in a subject. For example, if the baseline blood cells obtained from a subject are higher than the blood cells measured from the same subject after the C3 dsRNA agent or C3 antisense polynucleotide agent of the present invention is administered to the subject, it indicates the regression of the C3-related disease or condition and indicates the efficacy of the administered C3 dsRNA agent of the present invention in treating the C3-related disease or condition.

[0204] Some embodiments of the present invention include determining the presence, absence, and / or changes in physiological characteristics of a C3-related disease or condition using methods such as, but not limited to, (1) measuring blood cells in a subject; (2) evaluating physiological characteristics of one or more biological samples obtained from one or more subjects; or (3) performing a physical examination of a subject. This assay can be used to evaluate the efficacy of the treatment methods of the present invention.

[0205] medicine box

[0206] Kits comprising one or more C3 dsRNA agents and / or C3 antisense polynucleotide agents and instructions for use in the methods of the present invention are also within the scope of the present invention. The kits of the present invention may comprise one or more of a C3 dsRNA agent, a C3 sense polynucleotide, and a C3 antisense polynucleotide agent useful for treating a C3-related disease or condition. Kits comprising one or more C3 dsRNA agents, C3 sense polynucleotides, and C3 antisense polynucleotide agents may be prepared for use in the therapeutic methods of the present invention. The components of the kits of the present invention may be packaged in aqueous media or lyophilized form. The kits of the present invention may comprise a carrier that is partitioned to enclose one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) therein. The first container device or the series of container devices may comprise one or more compounds, such as a C3 dsRNA agent and / or a C3 sense or antisense polynucleotide agent. A second container means or series of container means may contain targeting agents, labeling agents, delivery agents, etc., which may be included as part of the C3 dsRNA agent and / or C3 antisense polynucleotide administered in embodiments of the treatment methods of the invention.

[0207] The kit of the present invention may also include instructions. The instructions are typically in written form and will provide guidance for performing the treatment embodied by the kit and for making decisions based on the treatment.

[0208] The following examples are provided to illustrate specific examples of the practice of the present invention and are not intended to limit the scope of the invention. It will be apparent to one of ordinary skill in the art that the present invention can be applied to a variety of compositions and methods.

[0209] Example

[0210] Example 1. Synthesis of RNAi Reagents

[0211] The C3 RNAi reagent duplexes shown in Tables 2-3 above were synthesized according to the following general procedure:

[0212] The sense and antisense strands of siRNA were synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. The oligonucleotide chain growth was achieved through a four-step cycle: deprotection, condensation, capping, and oxidation or sulfurization steps for each nucleotide addition. The synthesis was performed on a controlled pore glass (CPG) ). Monomeric phosphoramidites were purchased from commercial sources. Phosphoramidites with GalNAc ligand clusters (GLPA1, GLPA2, and GLPA15 as non-limiting examples) were synthesized according to the procedures of Examples 2-3 herein. For siRNAs used for in vitro screening (Table 2), synthesis was performed at a 2 μmol scale; for siRNAs used for in vivo testing (Table 3), synthesis was performed at a scale of 5 μmol or greater. In the case where the GalNAc ligand (GLO-0 as a non-limiting example) was attached to the 3'-end of the sense strand, a CPG solid support to which the GalNAc ligand was attached was used. In the case where the GalNAc ligand (GLS-1, GLS2, or GLS-15 as non-limiting examples) was attached to the 5'-end of the sense strand, the GalNAc phosphoramidite (GLPA1, GLPA2, or GLPA15 as non-limiting examples) was used for the final coupling reaction. 3% trichloroacetic acid (TCA) in dichloromethane was used for deprotection of the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole was used as an activating agent. I2 in THF / Py / H2O and phenylacetyl disulfide (PADS) in pyridine / MeCN were used for oxidation and sulfidation reactions, respectively. After the final solid-phase synthesis step, the solid support-bound oligomer was cleaved and the protecting groups removed by treatment with a 1:1 volume of 20 wt% aqueous methylamine and 28% ammonium hydroxide solution. To synthesize siRNA for in vitro screening, the crude mixture was concentrated. The remaining solid was dissolved in 1.0 M NaOAc, and ice-cold EtOH was added to precipitate the single-stranded product as a sodium salt, which was used for annealing without further purification. To synthesize siRNA for in vivo testing, the crude single-stranded product was further purified by ion-pair reversed-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving it in 1.0 M NaOAc and precipitating it by adding ice-cold EtOH. The sense and antisense oligonucleotides were annealed with equimolar complementarity in water to form a double-stranded siRNA product, which was lyophilized to provide a fluffy white solid.

[0213] Example 2. Preparation of Intermediate-A and Intermediate-B

[0214] As shown in Scheme 1 below, intermediate-A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). Glycosylation was then performed with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol to provide compound II. The Cbz protecting group was removed by hydrogenation to provide intermediate-A as a trifluoroacetate (TFA) salt. Intermediate B was synthesized based on the same protocol, except that Cbz-protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as the starting material.

[0215] Solution 1

[0216] To a solution of compound I (20.0 g, 51.4 mmol) in 100 mL of 1,2-dichloroethane (DCE) was added TMSOTf (17.1 g, 77.2 mmol). The resulting reaction solution was stirred at 60°C for 2 hours and then at 25°C for 1 hour; Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) was dissolved in DCE (100 mL) with Powdered molecular sieve (10g) is dried and added dropwise to the above-mentioned reaction solution at 0°C under N2 atmosphere.Under N2 atmosphere, the resulting reaction mixture is stirred at 25°C for 16 hours.The reaction mixture is filtered and washed with saturated NaHCO3 (200mL), water (200mL) and saturated brine (200mL).The organic layer is dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain a crude product, which is ground together with 2-methyltetrahydrofuran / heptane (5 / 3, v / v, 1.80L) for 2 hours.The resulting mixture is filtered and dried to obtain Compound II (15.0g, 50.3% yield) as a white solid.

[0217] 10% Pd / C (1.50 g) was carefully added to a dry and argon-purged hydrogenation bottle, followed by 10 ml of tetrahydrofuran (THF), followed by a solution of compound II (15.0 g, 26.4 mmol) in THF (300 ml) and TFA (trifluoroacetic acid, 3.00 g, 26.4 mmol). The resulting mixture was degassed and purged 3 times with H2 and stirred at 25 ° C for 3 hours under an atmosphere of H2 (45 psi). Thin layer chromatography (TLC, solvent: DCM: MeOH = 10: 1) showed that compound II was completely consumed. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. The process was repeated 3 times to obtain intermediate-A (14.0 g, 96.5% yield) as a foamy white solid. 1H NMR (400MHz DMSO-d6): δppm 7.90 (d, J=9.29Hz, 1H), 7.78 (br s, 3H), 5.23 (d, J = 3.26Hz, 1H), 4.98 (dd, J = 11.29, 3.26Hz, 1H), 4.56 (d, J = 8. 53Hz,1H),3.98-4.07(m,3H),3.79-3.93(m,2H),3.55-3.66(m,5H),2.98(br d,J=4.77Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.76(s,3H).

[0218] Intermediate-B was synthesized using a similar procedure to that used for Intermediate-A. 1H NMR (400 MHz DMSO-d6): δ ppm 7.90 (br d, J = 9.03 Hz, 4H), 5.21 (d, J = 3.51 Hz, 1H), 4.97 (dd, J = 11.1 Hz, 1H), 4.54 (d, J = 8.53 Hz, 1H), 3.98-4.06 (m, 3H), 3.88 (dt, J = 10.9 Hz, 1H), 3.76-3.83 (m, 1H), 3.49-3.61 (m, 9H), 2.97 (br s, 2H), 2.10(s, 3H), 1.99(s, 3H), 1.88(s, 3H), 1.78(s, 3H). Calculated mass spectrum C20H34N2O11: 478.22; found: 479.3(M+H+).

[0219] Example 3. Synthesis of GalNAc ligand cluster phosphoramidites GLPA1, GLPA2, and GLPA15.

[0220] GLPA1 and GLPA2 were prepared according to Scheme 2 below. Starting from benzyl-protected propane-1,3-diamine, it was alkylated with tert-butyl 2-bromoacetate to provide the triester compound I. The benzyl protecting group was removed by hydrogenation to provide the secondary amine compound II. The amide was coupled with 6-hydroxyhexanoic acid to provide compound III. The tert-butyl protecting group was then removed upon treatment with HCl in dioxane to generate the triacid compound IV. Amide coupling between triacid compound IV and intermediate-A or intermediate-B was performed to provide compound Va or Vb. The phosphoramidites GLPA1 or GLPA2 were synthesized by phosphitylation of compound Va or Vb with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.

[0221] Option 2

[0222] To a solution of N-benzyl-1,3-propanediamine (5.00 g, 30.4 mmol) in dimethylformamide (DMF, 100 mL) was added tert-butyl 2-bromoacetate (23.7 g, 121 mmol); diisopropylethylamine (DIEA, 23.61 g, 182 mmol) was then added dropwise. The resulting reaction mixture was stirred at 25-30°C for 16 hours. LCMS indicated complete consumption of N-benzyl-1,3-propanediamine. The reaction mixture was diluted with H₂O (500 mL) and extracted with EtOAc (500 mL x 2). The combined organics were washed with saturated brine (1 L), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to obtain the crude product, which was then purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 20:1 to 5:1). Compound I (12.1 g, 78.4% yield) was obtained as a colorless oil. 1H NMR (400MHz, CDCl3): δppm 7.26-7.40(m,5H),3.79(s,2H),3.43(s,4H),3.21(s,2H),2.72(dt,J=16.9,7.34Hz,4H),1.70(quin,J=7.2Hz,2H),1.44-1.50(m,27H).

[0223] The dried hydrogenation bottle was purged with argon 3 times. Pd / C (200 mg, 10%) was added, followed by MeOH (5 mL), followed by a solution of compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with H 2 . This process was repeated 3 times. The mixture was stirred at 25 ℃ for 12 hours under an H 2 (15 psi) atmosphere. LCMS showed that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under an N 2 atmosphere. The filtrate was concentrated under reduced pressure to obtain compound II (655 mg, 79.7% yield) as a yellow oil, which can be used in the next step without further purification. 1H NMR (400MHz, CDCl3): δppm 3.44 (s, 4H), 3.31 (s, 2H), 2.78 (t, J = 7.1Hz, 2H), 2.68 (t, J = 6.9Hz, 2H), 1.88 (br s, 1H), 1.69 (quin, J = 7.03Hz, 2H), 1.44-1.50 (s, 27H).

[0224] A mixture of compound II (655 mg, 1.57 mmol), 6-hydroxyhexanoic acid (249 mg, 1.89 mmol), DIEA (1.02 g, 7.86 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 904 mg, 4.72 mmol) and 1-hydroxybenzotriazole (HOBt, 637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged with N 2 three times; then stirred at 25 ° C. under an N 2 atmosphere for 3 hours. LCMS indicated the desired product. The reaction mixture was diluted with HO (10 mL) and extracted with EtOAc 20 mL (10 mL×2). The organics were combined and washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate from 5:1 to 1:1) to give Compound III (650 mg, 77.8% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ ppm 3.90-3.95 (s, 2H), 3.63 (t, J = 6.40 Hz, 2H), 3.38-3.45 (m, 6H), 2.72 (t, J = 6.65 Hz, 2H), 2.40 (t, J = 7.28 Hz, 2H), 1.55-1.75 (m, 8H), 1.44 (s, 27H). Mass spectrum calculated for C27H50N2O8: 530.36; found: 531.3 (M+H + ).

[0225] A mixture of compound III (5.5 g, 10.3 mmol) in HCl / dioxane (2 M, 55 mL) was stirred at 25 ° C for 3 hours. LCMS showed that compound III was completely consumed. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give a crude product. It was dissolved in CH3CN (50 mL) and volatiles were removed in vacuo. The process was repeated 3 times to obtain compound IV (2.05 g, 54.5% yield) as a white solid. 1 H NMR (400MHz, D2O): δppm 4.21(s,1H),4.07(d,J=4.5Hz,4H),3.99(s,1H),3.45-3.52(m,3H),3. 42(t,J=6.5Hz,1H),3.32-3.38(m,1H),3.24-3.31(m,1H),2.37(t,J=7 .4Hz,1H),2.24(t,J=7.4Hz,1H),1.99(dt,J=15.5,7.53Hz,1H),1.85-1.94(m,1H),1.85-1.94(m,1H),1.39-1.56(m,4H),1.19-1.31(m,2H).

[0226] A mixture of compound IV (500 mg, 1.05 mmol), intermediate-A (2.02 g, 3.67 mmol), DIEA (813 mg, 6.30 mmol), EDCI (704 mg, 3.67 mmol) and HOBt (496 mg, 3.67 mmol) in DMF (10 milliliters) was degassed and purged with N2 three times, and then the mixture was stirred at 25 ° C under N2 atmosphere for 3 hours. LCMS indicates the desired product. The reaction mixture was quenched by adding H2O (10 mL) and extracted with DCM (10 mL × 2). The combined organic matter was extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with saturated NaHCO3 solution and re-extracted with DCM (10 mL x 2). The organic matter was dried over sodium sulfate, filtered and concentrated under reduced pressure to produce compound Va (570 mg, 0.281 mmol, 26.8% yield) as a white solid. 1H NMR:(400MHz,CDCl3)ppmδ7.84-8.12(m,3H),6.85-7.15(m,2H),6.66-6.81(m,1H),5.36(br d,J=2.7Hz,3H),5.11-5.27(m,3H),4.63-4.85(m,3H),3.90-4.25(m,18H),3.37-3.75(m,28H),3.15-3.28(m,4H),2.64(br d,J=6.53Hz,2H),2.30-2.46(m,2H),2.13-2.18(m,9H),2.05(s,9H),1.94-2.03(m,18H),1.68(br s,2H),1.45(br s,2H),1.12(br t,J=7.0Hz,2H)。

[0227] To a solution of compound Va (260 mg, 0.161 mmol) in anhydrous DCM (5 mL) was added diisopropylammonium tetrazolium (30.3 mg, 0.177 mmol); then 3-bis(diisopropylamino)phosphinoyloxypropionitrile (194 mg, 0.645 mmol) was added dropwise at ambient temperature and N2. The reaction mixture was stirred at 20 to 25 ° C for 2 hours. LCMS showed that compound Va was completely consumed. After cooling to -20 ° C, the reaction mixture was added to a stirred brine / saturated NaHCO3 (1:1, 5 mL) solution at 0 ° C. After stirring for 1 minute, DCM (5 mL) was added. Layers separated. The organic layer was washed with brine / saturated NaHCO3 aqueous solution (1:1, 5 mL), dried over Na2SO4, filtered and concentrated to a volume of approximately 1 mL. The residual solution was added dropwise to 20 mL of methyl tert-butyl ether (MTBE) under stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged and the solid was collected. The solid was redissolved in 1 mL of DCM and precipitated by adding MTBE (20 mL). The solid was isolated again by centrifugation. The collected solid was dissolved in anhydrous CH3CN. The volatiles were removed. This process was repeated two more times to obtain the GalNAc ligand phosphoramidite compound GLPA1 (153 mg, 84.4 μmol) as a white solid. 1H NMR (400MHz, CDCl3): ppmδ7.71-8.06(m,2H),6.60-7.06(m,3H),5.37(br d,J=3.0Hz,3H),5.18-5.32(m,3H),4.70-4.86(m,3H),3.92-4.25(m,18H),3.42-3.85(m,30H),3.25(m ,4H),2.59-2.75(m,4H),2.27-2.44(m,2H),2.15-2.20(s,9H)2.07(s,9H),1.96-2.03(m,18H),1.65(br s,4H),1.44(br d,J=7.28Hz,2H),1.14-1.24(m,12H).31PNMR(CDCl3):ppmδ147.15.

[0228] The GalNAc ligand phosphoramidite compound GLPA2 was synthesized using the same procedure except that Intermediate-B was used. 1H NMR (400 MHz, CDCl 3 ): ppm δ 7.94-8.18 (m, 1H), 7.69 (br s,1H),6.66-7.10(m,3H),5.35(d,J=3.5Hz,3H),5.07-5.25(m,3H),4.76-4 .86(m,3H),4.01-4.31(m,10H),3.91-4.01(m,8H),3.74-3.86(m,4H),3.52- 3.71(m,30H),3.42-3.50(m,6H),3.15-3.25(m,4H),2.52-2.70(m,4H),2.2 2-2.45(m,2H),2.15-2.22(s,9H),2.06(s,9H),1.95-2.03(m,18H),1.77(br s,2H),1.58-1.66(m,4H),1.40(m,2H),1.08-1.24(m,12H).31PNMR(CDCl3):ppmδ147.12.

[0229] GLPA15 was prepared according to the following protocol 3.

[0230] To a solution of intermediate compound II (275 g, 660 mmol, 1.00 equivalent (eq)) in dichloromethane (2.75 L) was added triethylamine (133 g, 1.32 mol, 2.00 eq.), followed by the dropwise addition of Cbz-Cl (169 g, 990 mmol, 1.50 eq.). The reaction mixture was stirred at 25°C for 2 hours. LCMS indicated complete conversion of compound II. The reaction mixture was washed sequentially with a saturated solution of NaHCO₃ (800 mL) and then saturated brine (500 mL). The organic phase was dried over anhydrous Na₂SO₄. After filtering to remove the desiccant, the filtrate was concentrated to dryness. The crude product was purified by column chromatography (SiO₂, PE / EA = 100 / 1 to 5 / 1) to afford compound III-1 (290 g, 527 mmol, 75.7% yield) as a colorless oil. 1 H NMR (400MHz in DMSO-d6): δppm 7.23-7.40(m,5H),5.00-5.12(m,2H),3.86-3.95(m,2H),3.23-3.39(m,6H ),2.55-2.67(m,2H),1.56-1.64(m,2H),1.31-1.46(m,27H).MS(ESI)[M+H] + m / z:551.6.

[0231] To compound III-1 (145 g, 263 mmol, 1.00 eq) was added HCOOH (2.9 L), and the solution was stirred at 60°C for 12 hours. LCMS showed complete conversion of compound III-1. 1.5 L of toluene and 1.5 L of acetonitrile were added to the reaction solution, which was concentrated under reduced pressure to approximately 500 mL. Toluene / acetonitrile (1:1, ~750 mL) was then added and concentrated to approximately 500 mL. Acetonitrile (~1000 mL) was then added and concentrated to dryness. The crude product was triturated with 700 mL of acetonitrile at 60°C for 2 hours and filtered. The solid was collected and dried to yield compound IV-1 (105 g, quantitative) as a white solid. 1H NMR (400MHz in DMSO-d6): δppm 7.26-7.40(m,5H),5.02-5.10(m,2H),3.89-4.00(m,2H),3.36-3.45(m,4H),3.24-3.34(m,2H),2.59-2.72(m,2H),1.40(s,2H).MS(ESI)[M+H] + m / z:383.0.

[0232] To a solution of compound IV-1 (100 g, 261 mmol) and intermediate-A (502 g, 915 mmol, 3.50 eq.) in DMF (1.0 L) were added TBTU (327 g, 1.02 mol, 3.90 eq.) and triethylamine (212 g, 2.09 mol, 8.00 eq.). The reaction was allowed to proceed at 25°C for 1 hour. LCMS indicated complete conversion of compound IV-1. The reaction solution was added to 4000 mL of water and extracted with methyl tert-butyl ether (2000 mL in two portions) to remove impurities. The remaining aqueous phase was extracted with dichloromethane (3000 mL in two portions). The dichloromethane phase was washed sequentially with 10% aqueous citric acid (2000 mL in two portions), saturated NaHCO₃ (2.0 L in two portions), and saturated brine (2.0 L), and then dried over anhydrous Na₂SO₄. The filtrate was filtered and concentrated under reduced pressure to give white solid compound V-1 (260 g, 159 mmol, yield 60.9%). 1H NMR (400MHz in DMSO-d6): δppm 7.99-8.08(m,2H),7.93(br d,J=5.50Hz,1H),7.79-7.86(m,3H),7.26-7.39(m,5H),5.22(d,J=3.13Hz,3H),4.95-5.08(m,5H),4.54(br d,J=8.38Hz,3H),4.03(s,9H),3.81-3.93(m,5H),3.76(br d,J=4.88Hz,3H),3.44-3.62(m,10H),3.34-3.43(m,6H),3.24(br d,J=6.13Hz,7H),3.02-3.09(m,4H),2.40-2.47(m,2H),2.10(s,9H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.57-1.68(m,2H). MS(ESI)[M+H] + m / z:816.4.

[0233] A 2L hydrogenation reactor was inertized with argon and dry Pd / C (9 g) was carefully added. MeOH (50 mL) was then added to moisten the Pd / C. Then, under an argon atmosphere, a solution of compound V-1 (90 g, 55.1 mmol, 1.00 eq.) and trifluoroacetic acid (6.29 g, 55.1 mmol, 1.00 eq.) in MeOH (850 mL) was slowly added. The mixture was degassed and replaced with a hydrogen atmosphere by adding H2 three times, and stirred at 25°C for 10 hours. LCMS indicated complete conversion of compound V-1. The Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to afford compound VI-1 (80 g, 90.2% yield). 1H NMR (400MHz in DMSO-d6): δppm 9.12 (br s, 2H), 8.50 (br t, J=5.19Hz, 1H), 8.10 (br t,J=5.50Hz,2H),7.85-7.91(m,3H),5.22(d,J=3.25Hz,3H),4.95-5.01(m,3H),4.52 -4.58(m,3H),4.03(s,9H),3.84-3.93(m,3H),3.75-3.83(m,3H),3.39-3.61(m,16H) ,3.23-3.32(m,6H),3.15-3.18(m,3H),2.97-3.05(m,2H),2.54-2.61(m,2H),2.10(s ,9H),2.00(s,9H),1.89(s,9H),1.77-1.80(m,9H),1.70-1.76(m,2H).MS(ESI)[M+H] + m / z:749.3.

[0234] To a solution of compound VI-1 (270 g, 168 mmol, 1.00 eq.) and glutaric anhydride (28.6 g, 252 mmol, 1.50 eq.) in dichloromethane (2.7 L) was added triethylamine (67.8 g, 672 mmol, 4.00 eq.). The solution was stirred at 25°C for 1 hour. LCMS indicated complete conversion of compound VI-1 to compound VII. 4-Hydroxypiperidine (42.4 g, 420 mmol, 2.50 eq.) and TBTU (107 g, 335 mmol, 2.00 eq.) were added to the reaction mixture, and stirring was continued at 25°C for 1 hour. LCMS indicated complete conversion of compound VII. The reaction was quenched by the slow addition of saturated NH4Cl (3.0 L). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 1000 mL) and combined with the previous organic phase. The combined organic phases were washed with a 1:1 mixture of saturated NaHCO₃(aq) and saturated brine (3.0 L), dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was dissolved in 1.5 L of dichloromethane and added dropwise to methyl tert-butyl ether (7.5 L). A translucent white precipitate gradually formed during the addition. The precipitate was vacuum filtered, and the collected solid was dried under vacuum to yield Compound VIII as a white solid (207 g, 72.8% yield). 1H NMR (400MHz in DMSO-d6): δppm 8.05(br d,J=2.00Hz,2H),7.82(br d,J=7.38Hz,3H),5.21(br s,3H),4.98(br d,J=10.26Hz,3H),4.72(br s,1H),4.54(br d,J=7.88Hz,3H),4.03(br s,9H),3.74-3.94(m,9H),3.45-3.71(m,12H),3.40(br s,6H),3.24(br s,7H),3.07(br d,J=14.13Hz,5H),2.91-3.01(m,1H),2.24-2.44(m,5H),2.20(br s,1H),2.10(s,9H),1.96-2.04(m,9H),1.89(br s,9H),1.74-1.81(m,9H),1.51-1.73(m,6H),1.07-1.36(m,3H).MS(ESI)[M+H] + m / z:848.0.

[0235] To a solution of compound VIII (200 g, 118 mmol, 1.00 eq.) and diisopropylammonium tetrazolium (8.08 g, 47.2 mmol, 0.40 eq) in dichloromethane (2.0 L) was added 3-bis(diisopropylamino)phosphonooxypropionitrile (53.3 g, 177 mmol, 1.50 eq.). The reaction solution was stirred at 40 °C for 2 hours. LCMS showed that the conversion of compound 13 was complete. The reaction mixture was washed with a 1:1 mixture of saturated NaHCO₃ and saturated brine (2.0 L) and dried over anhydrous Na₂SO₄. The filtrate was concentrated, and the resulting crude product was dissolved in dichloromethane (1.2 L) and added dropwise to stirred methyl tert-butyl ether (6.0 L). The suspension was filtered, and the filter cake was rinsed with methyl tert-butyl ether. The collected solid was vacuum-dried, dissolved in dichloromethane (1.0 L), and concentrated to dryness. This process was repeated four times to remove any residual tert-butyl ether, yielding GLPA15 (164 g, 73.3% yield). 1H NMR (400MHz in DMSO-d6): δppm 8.05(br d,J=6.50Hz,2H),7.81(br d,J=9.01Hz,3H),5.22(d,J=3.25Hz,3H),4.98(dd,J=11.26,3.25Hz,3H),4.55(br d,J=8.50Hz,3H),4.03(s,9H),3.64-3.97(m,12H),3.55-3.63(m,6H),3.50(br s,5H),3.40(br d,J=6.13Hz,6H),3.17-3.30 (m,9H),3.07(br d,J=14.26Hz,4H),2.76(t,J=5.82Hz,2H),2.18-2.47(m,6H),2.10(s,9H),1.9 9(s,9H),1.89(s,9H),1.78(s,9H),1.52-1.74(m,6H),1.12-1.19(m,12H).31P NMR(DMSO-d6):ppmδ145.25.MS(ESI)[M+H] + m / z:1895.7.

[0236] In certain studies, a method is provided for attaching a targeting group comprising GalNAc (also referred to herein as a GalNAc delivery compound) to the 5'-end of the sense strand, which comprises using a GalNAc phosphoramidite (GLPA1) in the last coupling step of solid phase synthesis, using such a synthesis process, such as that used in oligonucleotide chain extension (i.e., adding nucleotides to the 5' end of the sense strand), to attach it to the 5'-end of the sense strand.

[0237] In some studies, a method for attaching a targeting group comprising GalNAc to the 3'-end of the sense strand includes using a solid support (CPG) comprising GLO-n. In some studies, a method for attaching a targeting group comprising GalNAc to the 3'-end of the sense strand includes attaching the GalNAc targeting group to a CPG solid support via an ester bond, and using the resulting CPG with the attached GalNAc targeting group when synthesizing the sense strand, which results in the GalNAc targeting group being attached to the 3'-end of the sense strand. Other GalNAc phosphoramidite compounds (GLPAn) can also be obtained using similar methods herein or well known in the art after using reasonably corresponding intermediates, and attached as targeting groups to appropriate positions of the siRNA duplex.

[0238] Example 4. In vitro screening of C3 siRNA duplexes

[0239] Huh7 cells were trypsinized and adjusted to the appropriate density before being seeded into 96-well plates. Simultaneously with seeding, cells were transfected with test siRNAs or PBS controls using Lipofectamine RNAiMax (Invitrogen -13778-150) according to the manufacturer's recommendations. siRNAs were tested at two concentrations (0.08 nM and 0.6 nM) in triplicate.

[0240] 24 hours after transfection, the culture medium was removed and the cells were harvested for RNA extraction. TRIzol was used according to the manufacturer's instructions. TM Total RNA was extracted using ELISA Reagent (Invitrogen-15596018).

[0241] According to the manual, use PrimeScript TM cDNA was synthesized using an RT kit and gDNA Eraser (Perfect Real Time) (TaKaRa-RR047A). C3 cDNA was detected by qPCR. GAPDH cDNA was detected in parallel as an internal control. PCR was performed as follows: 95°C for 30 seconds, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds.

[0242] Data Analysis

[0243] The expression of the C3 gene in each sample was determined by relative quantification (RQ) using the comparative Ct (ΔΔCt) method; this method measures the Ct difference (ΔCt) between the target gene and the housekeeping gene (GAPDH).

[0244] The equation is listed as follows: ΔCT = target gene average Ct - GAPDH average Ct ΔΔCT = ΔCT (sample) - ΔCT (random control or Lipofectamine RNAiMax control); Relative quantification of target gene mRNA = 2^(-ΔΔCT) Inhibition % = (relative quantification of control - relative quantification of sample) / relative quantification of control × 100%.

[0245] Table 4 provides experimental results from in vitro studies using various C3 RNAi agents to inhibit C3 expression; the double-stranded sequences used correspond to the compounds shown in Table 2.

[0246] Example 5. In vivo testing of C3 siRNA duplexes

[0247] To evaluate the in vivo activity of C3 siRNA, mice infected with AAV encoding human C3 and luciferase genes were used (4 mice per group). 14 days before siRNA administration, female C57BL / 6J mice were infected by intravenous injection of 1x10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human C3 and luciferase genes. On day 0, mice were subcutaneously injected with a single dose of 3 mg / kg or 6 mg / kg of C3 siRNA reagent or PBS. Blood samples were collected before siRNA administration on day 0 and at the end of days 7 and 14. Luciferase activity was measured. By comparing the luciferase activity of the blood samples before administration of the siRNA treatment group and the luciferase activity of the blood samples collected on days 7 and 14 and normalized based on the changes in luciferase activity in the serum samples from the PBS treatment group, the remaining percentage was calculated. The results are shown in Table 5.

[0248] Table 5. Compounds using sequences, chemical modifications, and delivery methods corresponding to those shown in Table 3. GLX-n is the delivery compound represented by GalNAc3 in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961.

[0249] Example 6. In vivo testing of C3 siRNA duplexes

[0250] To evaluate the in vivo activity of C3 siRNA, mice infected with AAV encoding human C3 and luciferase genes were used (4 mice per group). Seven days before siRNA administration, female C57BL / 6J mice were infected by intravenous injection of 1x10^11viral particles of adeno-associated virus 8 (AAV8) vector encoding human C3 and luciferase genes. On day 0, mice were subcutaneously injected with a single dose of 3 mg / kg of C3 siRNA reagent or PBS. Blood samples were collected before siRNA administration on day 0 and at the end of day 7. Blood samples were collected on day 7 in the siRNA-treated group and the PBS-treated group, and the concentration of human C3 protein in the plasma samples normalized to the changes before treatment was measured by ELISA to calculate the retention percentage; the results are shown in Tables 6-8.

[0251] Table 6. Compounds using sequences, chemical modifications, and delivery methods corresponding to those shown in Table 3. GLX-n is the delivery compound represented by GalNAc3 in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961.

[0252] Table 7. Compounds corresponding to the sequences, chemical modifications, and delivery methods shown in Table 3 were used at 3 mg / kg each. GLX-n is the delivery compound represented by GalNAc3 in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961.

[0253] Table 8. Compounds corresponding to the sequences, chemical modifications, and delivery methods shown in Table 3 were used at 3 mg / kg each. GLX-n is the delivery compound shown as GalNAc3 in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958-16961.

[0254] Example 7. In vivo testing of C3 siRNA duplexes

[0255] To evaluate the in vivo activity of C3 siRNA, mice infected with AAV encoding human C3 and luciferase genes were used (4 mice per group). Seven days before siRNA administration, female C57BL / 6J mice were infected by intravenous injection of 1x10^11 viral particles of adeno-associated virus 8 (AAV8) vector encoding human C3 and luciferase genes. On day 0, mice were subcutaneously injected with a single dose of 3 mg / kg or 6 mg / kg of C3 siRNA reagent or PBS. Blood samples were collected before siRNA administration on day 0 and at the end of days 7, 14, and 21. Blood samples were collected on days 7, 14, and 21 in the siRNA-treated group and the PBS-treated group. The concentration of human C3 protein in the plasma samples was normalized relative to the change before treatment by ELISA to calculate the retention percentage; the results are shown in Tables 9-11.

[0256] Table 9. Percent retention of human C3 in mouse plasma measured by ELISA for compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 3, using a single 3 mpk dose.

[0257] Table 10. Percent retention of human C3 in mouse plasma measured by ELISA for compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 3, using a single 6 mpk dose.

[0258] Table 11. Percent retention of human C3 in mouse plasma measured by ELISA following a single 6 mpk dose of compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 3.

[0259] equivalent

[0260] Although several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily appreciate that a variety of other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantages, as well as each of these variations and / or modifications, are considered to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and / or configurations will depend on the specific application in which the teachings of the present invention are used. Those skilled in the art will recognize or be able to ascertain, using only routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented by way of example only and are within the scope of the appended claims and their equivalents, and that the present invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and / or methods, provided that such features, systems, articles, materials, and / or methods are not mutually inconsistent, is also within the scope of the present invention.

[0261] All definitions, as defined and used herein, should be understood against dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0262] Where no quantity limitation is used in the specification and claims, it should be understood as "at least one" unless explicitly stated to the contrary.

[0263] The phrase "and / or" as used in the specification and claims should be understood to mean "one or both" of the elements so conjoined, i.e., such elements are present in combination in some cases and separately in other cases. In addition to the elements specifically identified by "and / or," other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless explicitly stated to the contrary.

[0264] All references, patents and patent applications and publications cited or referenced in this application are hereby incorporated by reference in their entirety.

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C3, wherein the dsRNA agent comprises a sense strand and an antisense strand, nucleotides 2 to 18 of the antisense strand comprise a region complementary to a C3 RNA transcript, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the antisense sequences listed in one of Tables 1-3 by 0, 1, 2 or 3 nucleotides, and optionally comprises a targeting ligand.

2. The dsRNA agent of claim 1, wherein the region complementary to the C3 RNA transcript comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3.

3. The dsRNA agent of claim 1 or 2, wherein the antisense strand of the dsRNA is at least substantially complementary to any one of the target regions in SEQ ID NO: 1 and provided in any one of Tables 1-3.

4. The dsRNA agent of claim 3, wherein the antisense strand of the dsRNA is fully complementary to any target region in SEQ ID NO: 1 and provided in any one of Tables 1-3.

5. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sense strand sequence of any one of Tables 1-3, wherein the sense strand sequence is at least substantially complementary to an antisense strand sequence in the dsRNA agent.

6. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sense strand sequence of any one of Tables 1-3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent.

7. The dsRNA agent of claim 1, wherein the dsRNA agent comprises an antisense strand sequence listed in any one of Tables 1-3.

8. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sequence listed as a duplex sequence in any one of Tables 1-3.

9. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one modified nucleotide.

10. The dsRNA agent of claim 1, wherein all or substantially all nucleotides in the antisense strand are modified nucleotides.

11. The dsRNA agent of claim 9 or 10, wherein the at least one modified nucleotide comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seco nucleotide mimetics, locked nucleotides, open ring nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted abasic nucleotides, inverted 2'-OMe nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides containing a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base containing nucleotide.

12. The dsRNA agent of claim 9 or 10, wherein the guide strand comprises an E-vinylphosphonate nucleotide at the 5' end.

13. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one phosphorothioate internucleoside linkage.

14. The dsRNA agent of claim 1, wherein the sense strand comprises at least one phosphorothioate internucleoside linkage.

15. The dsRNA agent of claim 1, wherein the antisense strand comprises at least one phosphorothioate internucleoside linkage.

16. The dsRNA agent of claim 1, wherein the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

17. The dsRNA agent of claim 1, wherein the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

18. The dsRNA agent of claim 1, wherein all or substantially all nucleotides of the sense and antisense strands are modified nucleotides.

19. The dsRNA agent of claim 1, wherein the modified sense strand is a modified sense strand sequence listed in one of Tables 2-3.

20. The dsRNA agent of claim 1, wherein the modified antisense strand is a modified antisense strand sequence listed in one of Tables 2-3.

21. The dsRNA agent of claim 1, wherein the sense strand is complementary or substantially complementary to the antisense strand, and the complementary region is 16 to 23 nucleotides in length.

22. The dsRNA agent of claim 21, wherein the region of complementarity is 19 to 21 nucleotides in length.

23. The dsRNA agent of claim 1, wherein each strand is no more than 30 nucleotides in length.

24. The dsRNA agent of claim 1, wherein each strand is no more than 25 nucleotides in length.

25. The dsRNA agent of claim 1, wherein each strand is no more than 23 nucleotides in length.

26. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups.

27. The dsRNA agent of claim 26, wherein the one or more targeting groups or linking groups are conjugated to the sense strand.

28. The dsRNA agent of claim 26 or 27, wherein the targeting group or linker group comprises N-acetyl-galactosamine (GalNAc).

29. The dsRNA agent of claim 28, wherein the targeting group or linking group has a structure as shown in formula (X), comprising a targeting moiety, a linker, and a linker W, wherein the targeting moiety is selected from an N-acetyl-galactosamine derivative (GalNAc), which is linked to the linker W via a linker, and the linker W has a structure as shown in formula (XI), wherein X is selected from O, NH2 or S, and Y ﹣ Selected from: O ﹣ 、S ﹣ , methyl or NRaRb, Ra and Rb are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, or Ra and Rb together with the attached atoms are linked to form a 3-12 membered heterocycloalkyl containing 1-3 N, O, S heteroatoms; preferably, in the substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, the substituent is selected from hydroxyl and amino; 30. The dsRNA agent of claim 29, wherein the linker in the targeting group is selected from polyethylene glycol, optionally substituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 Amide.

31. The dsRNA agent of claim 30, wherein the substituted or unsubstituted C2-C 12 Alkyl, substituted or unsubstituted C3-C 12 Cycloalkyl, substituted or unsubstituted C3-C 12 Heterocycloalkyl, substituted or unsubstituted C3-C 12 In the amide, the substituent is selected from hydroxyl and carbonyl.

32. The dsRNA agent of claim 30, wherein the linker in the targeting group is preferably selected from the following fragments: Each m is an integer from 1 to 6, each n, o, and p is 0 or 1, and each q is an integer from 1 to 6. 1 With q 2 Each independently is 0, 1 or 2.

33. The dsRNA agent of claim 32, wherein the linker in the targeting group is more preferably selected from the following fragments:

34. The dsRNA agent of claim 29, wherein the targeting portion of the targeting group has the following structural fragment: n' is 1 or 2.

35. The dsRNA agent of claim 29, wherein X is selected from O or S, and Y ﹣ Selected from: O ﹣ or S ﹣ .

36. The dsRNA agent of claim 26 or 27, wherein the targeting group has the following structure:

37. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a targeting group conjugated to the 5'-end of the sense strand.

38. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a targeting group conjugated to the 3'-end of the sense strand.

39. The dsRNA agent of claim 1, wherein the antisense strand comprises an inverted abasic residue at the 3'-end.

40. The dsRNA agent of claim 1, wherein the sense strand comprises one or two inverted abasic residues at the 3' or / and 5' end.

41. The dsRNA agent of claim 1, wherein the dsRNA agent has two blunt ends.

42. The dsRNA agent of claim 1, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

43. The dsRNA agent of claim 1, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

44. The dsRNA agent of claim 1, wherein the sense strand has an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3'-end and / or the 5'-end.

45. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C3, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein each strand is 14 to 30 nucleotides in length, comprising a region of complementarity to a C3 RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region comprises at least 15 consecutive nucleotides that differ from one of the antisense sequences set forth in Formula I by 0, 1, 2, or 3 nucleotides, and optionally comprises a targeting ligand, the antisense strand of the double-stranded ribonucleic acid (dsRNA) agent: <h2 style=";text-align:left;direction:ltr">5'-N<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> -(A1A2A3A4)iN<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> -(B1B2B3B4)-N<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> -(C1C2C3C4C5)jN<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> -3' Wherein: i and j are independently selected from 0 or 1; Each N a and N b Each independently represents 0-17 oligonucleotides; A1A2A3A4 represents a motif in which four consecutive nucleotides are modified by lowercase 2'-OMe, uppercase 2'-fluorine, lowercase 2'-OMe, and lowercase 2'-OMe, respectively, and further comprises phosphorothioate internucleoside linkages between A1 and A2, and between A2 and A3; B1B2B3B4 represents a motif in which four consecutive nucleotides are modified with lowercase 2′-OMe; C1C2C3C4C5 represents a motif in which five consecutive nucleotides are modified with lowercase 2'-OMe. The C4 and C5 further comprise phosphorothioate internucleoside linkages.

46. ​​The dsRNA agent of claim 45, N a Each independently represents 0 oligonucleotide, N b Each independently represents 2-5 chemically modified oligonucleotides, and i and j represent 1 respectively.

47. The dsRNA agent of claim 45, wherein the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO:

1.

48. The dsRNA agent of claim 45, wherein the antisense strand of the dsRNA is completely complementary to any target region of SEQ ID NO:

1.

49. The dsRNA agent of claim 45, wherein the sense strand sequence of any one of the dsRNAs is at least substantially complementary to the antisense strand sequence of the dsRNA agent.

50. The dsRNA agent of claim 45, wherein the sense strand sequence of any one of the dsRNAs is completely complementary to the antisense strand sequence of the dsRNA agent.

51. The dsRNA agent of claim 45, wherein the dsRNA sense strand comprises one or two inverted abasic residues at the 3' or / and 5' end.

52. The dsRNA agent of claim 45, wherein the dsRNA has two blunt ends.

53. The dsRNA agent of claim 45, wherein the dsRNA sense strand has an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3'-end and / or the 5'-end.

54. A composition comprising the dsRNA agent of any one of claims 1 to 53.

55. The composition of claim 54, further comprising a pharmaceutically acceptable carrier.

56. The composition of claim 55, further comprising one or more additional therapeutic agents.

57. The composition of claim 56, wherein the composition is packaged in a kit, container, package, dispenser, prefilled syringe, or vial.

58. The composition of claim 54, wherein the composition is formulated for subcutaneous administration or is formulated for intravenous (IV) administration.

59. A cell comprising the dsRNA agent of any one of claims 1 to 53.

60. The cell of claim 59, wherein The cell is a mammalian cell, optionally a human cell.

61. A method for inhibiting complement component C3 gene expression in a cell, comprising: (i) preparing a cell comprising an effective amount of a double-stranded ribonucleic acid (dsRNA) agent according to any one of claims 1 to 53 or a composition according to any one of claims 54 to 58; (ii) maintaining the cells prepared in (i) for a sufficient period of time to achieve degradation of the mRNA transcript of the C3 gene, thereby inhibiting the expression of the C3 gene in the cells.

62. The method of claim 61, wherein The cells are in a subject and the dsRNA agent is administered subcutaneously to the subject.

63. A method of inhibiting C3 gene expression in a subject, the method comprising administering to the subject an effective amount of the double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1 to 53 or the composition of any one of claims 54 to 58.

64. A method for treating a disease or condition associated with the C3 protein, the method comprising administering to a subject an effective amount of the double-stranded ribonucleic acid (dsRNA) agent of any one of claims 1 to 53 or the composition of any one of claims 54 to 58 to inhibit C3 gene expression.

65. The method of claim 64, wherein the disease or condition is caused by or associated with C3 activation or a symptom or progression thereof in response to C3 activation, the disease or condition being generally associated with C3 expression, the disease comprising one or more of: C3 glomerulopathy (C3G), immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus, lupus nephritis, ischemia / reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), primary membranous nephropathy, macular degeneration (AMD), rheumatoid arthritis (RA), anti-neutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegenerative diseases.

66. The method of claim 65, further comprising administering an additional therapeutic regimen to the subject.

67. The method of claim 66, wherein the additional treatment regimen comprises: One or more C3 antisense polynucleotides are administered to the subject, and a non-C3 dsRNA therapeutic agent is administered to the subject.

68. The method of claim 67, wherein the non-C3 dsRNA therapeutic agent is one or more of the following: an additional therapeutic agent, such as an anti-complement component C5 antibody or antigen-binding fragment thereof, an iRNA targeting complement component C5 and / or a C3 peptide inhibitor.

69. The method of claim 68, wherein the non-C3 dsRNA therapeutic agent is selected from one or more of the following: eculizumab or compulsinvastatin.