TW202405170A - Composition and method for inhibiting expression of complement component C3 protein - Google Patents

Abstract

The present invention provides a composition and method for use in inhibiting the expression of a complement component C3 gene and for treating C3-related diseases and disorders. Specifically, the present invention provides a dsRNA reagent, an antisense polynucleotide reagent, a composition containing the dsRNA reagent and a composition containing the antisense polynucleotide reagent, which can be used for reducing C3 gene expression in cells and in a target.

Description

Compositions and methods for inhibiting expression of complement component C3 protein

Some embodiments of the present invention relate to compositions and methods useful for inhibiting expression of complement component C3 protein.

Complement was first discovered in 1890 and is part of the innate immune system that protects the host from pathogen invasion. It is the first line of immune defense against foreign pathogens. Activating complement will lead to a series of sequential enzymatic reactions called complement activation, leading to the formation of the potent anaphylatoxins C3a and C5a, thereby triggering a plethora of physiological reactions ranging from chemical attraction to cell death. Complement was initially thought to play an important role in innate immunity to invading pathogens, however, more and more recent evidence has revealed that complement also plays an important role in adaptive immunity involving T cells and B cells, helping to eliminate pathogens (Dunkelberger JR and Song WC. (2010) Cell Res. 20:34; Molina H, et al. (1996) Proc Natl Acad Sci U S A. 93:3357); maintaining immunogenicity to avoid 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). Most of the components that form the complement system are proteins, including the complement component protein C3 (also referred to as C3 in this article), which are synthesized in large quantities and secreted into the blood in hepatocytes in the liver, activating the inflammatory response of the system leading to the attraction and opsonization of phagocytes function to remove pathogens, immune complexes, and cellular debris.

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 lectin 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 the primary complement pathway effector , one of the key components of 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.

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 glomerulonephritis) 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 C3-related diseases of other organs, such as neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), primary membranous nephropathy, are also known to be related to complement function Disorders involving other organs, such as age-related macular degeneration (AMD), rheumatoid arthritis (RA), antineutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease, and periodontal disease disease, malarial anemia, sepsis, and neurodegenerative diseases.

Currently, treatments are available for the treatment of complement component C3-related diseases that require time-consuming and invasive management, are expensive, and have a huge clinical unmet need; therefore, the field needs to give patients with complement component C3-related diseases an alternative therapy or combination therapy.

According to one aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) reagent for inhibiting the expression of complement component C3 is provided. The dsRNA reagent includes a sense strand and an antisense strand, and the nucleotide positions 2 to 3 in the antisense strand are 18 contains a region complementary to the C3 RNA transcript, where the complementary region contains at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in Tables 1-3 acid, and optionally includes a targeting ligand.

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

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

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

In some embodiments, the dsRNA reagent includes 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 reagent.

In some embodiments, the dsRNA reagent includes any one of the sense strand sequences listed in Tables 1-3, wherein the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA reagent.

In some embodiments, the dsRNA reagent comprises any of the antisense strand sequences listed in Tables 1-3.

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

In some embodiments, the dsRNA agent contains at least one modified nucleotide.

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 includes: 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-seco core Glycolic acid mimetic, locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), 2'-F-arabinose nucleoside acid, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, reverse nucleotide, reverse abasic nucleotide, reverse 2'-OMe nucleotide, reverse To 2'-deoxynucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, including 5'-sulfide Nucleotides with phosphoric acid ester groups, or terminal nucleotides linked to cholesterol derivatives or dodecyl dodecylamine groups, 2'-amino modified nucleotides, phosphoramidates or nucleosides containing Unnatural base of acid. In some embodiments, the antisense strand contains 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, of which less than 6 are 2' -Fluoronucleotide modified nucleotides. In certain embodiments, the antisense strand contains 3 or 5 2'-fluoronucleotides, preferably the antisense strand contains 5 2'-fluoronucleotides. In some embodiments, the sense strand contains 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, of which less than 4 are 2'- Fluoronucleotide modified nucleotides. In certain embodiments, the sense strand contains 3 2'-fluoronucleotides. In some embodiments, the antisense strand contains 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, wherein at least 16 modified nucleosides The acid is a 2'-O-methyl nucleotide and positions 2, 7, 12, 14 and/or 16 at the 5' end of the antisense strand are 2'-fluoronucleotide modified nucleotides (from the antisense strand) Counting begins with the first paired nucleotide at 5' of the strand). In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoronucleotides, wherein at least 18 modified nucleotides are 2'-O-methyl nucleotides and positions 9, 11 and/or 13 located at the 3' end of the sense strand are 2'-fluoronucleotide modified nucleotides (the first paired nucleotide from the 3' end of the sense strand counting from nucleotides). In some embodiments, the antisense strand is comprised in the direction from the 5' end to the 3' end, and nucleotides at positions 2, 7, 12, 14, and 16 of the antisense strand are 2'-fluoro modified cores The nucleotides are counted starting from the first paired nucleotide at the 5' end of the antisense strand, and nucleotides at other positions in each antisense strand are independently non-fluorine modified nucleotides. In some embodiments, the antisense strand includes 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'-fluoro modified nucleotides, from Counting begins with the first paired nucleotide 5' of the antisense strand, and each nucleotide elsewhere in the antisense strand is independently a non-fluorine modified nucleotide. In some embodiments, the sense strand contains nucleotides at positions 9, 11, and 13 of the sense strand in the direction from the 3' end to the 5' end, which are 2'-fluoro modified nucleotides, from the sense strand Counting begins with the first paired nucleotide at the 3' end, and each nucleotide elsewhere in the sense strand is independently a non-fluorine modified nucleotide. In some embodiments, the antisense strand includes a position in the direction from the 5' end to the 3' end of the antisense strand that is a UNA modified nucleotide, 5' from the first paired core of the antisense strand Start counting nucleotides.

In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of complement component C3, the dsRNA agent comprising a sense strand and an antisense strand, comprising at nucleotide positions 2 to 18 in the antisense strand A region complementary to the C3 RNA transcript, with the antisense strand being fully or partially complementary to the sense strand and optionally containing a targeting ligand, where each strand is 17 to 40 nucleotides in length, where the sense strand sequence contains Expressed by 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) where: each N′ _F represents a 2′-fluorine 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.

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

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

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.

In some embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the complementary region is between 16 and 23 nucleotides in length. In some embodiments, the complementary region is 19-21 nucleotides in length. In some embodiments, the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, each strand in the dsRNA agent is no more than 40 nucleotides in length. In some embodiments, each strand is no more than 30 nucleotides in length. In some embodiments, each strand is no more than 25 nucleotides in length. In some embodiments, each strand is no more than 23 nucleotides in length. In some embodiments, each strand is no more than 21 nucleotides in length. In some embodiments, each strand is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.

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

In some embodiments, a dsRNA agent contains at least one modified nucleotide and further contains one or more targeting 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 includes N-acetyl-galactosamine (GalNAc).

In some embodiments, the targeting group or linking group is a structure represented by formula (X), including a targeting moiety, a linker, and a linker W, wherein the targeting moiety is selected from N-acetyl-galactosamine Derivative (GalNAc), which is linked to the linker W through a linking bond. The linker W has a structure shown in formula (XI), X is selected from O, NH ₂ or S, Y ^- is selected from: O ^- , S ^- , A group or NRaRb, Ra and Rb are independently selected from hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, or Ra and Rb are with attached The atoms are linked together to form a 3-12 membered heterocycloalkyl group containing 1-3 N, O, and S heteroatoms. Formula (X) (Formula XI).

In some preferred embodiments, in the substituted or unsubstituted C ₁ -C ₆ alkyl group or the substituted or unsubstituted C ₃ -C ₆ cycloalkyl group, the substituent is selected from hydroxyl and amino. In some embodiments, X is selected from O or S, and Y ^- is selected from: O ^- , S ^- . In some embodiments, the linkage in the targeting group is selected from polyethylene glycol, optionally substituted C ₂ -C ₁₂ alkyl, substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl, substituted or unsubstituted Substituted C ₃ -C ₁₂ heterocycloalkyl, substituted or unsubstituted C ₃ -C ₁₂ amide. In some embodiments, substituted or unsubstituted C ₂ -C ₁₂ alkyl, substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl, substituted or unsubstituted C ₃ -C ₁₂ heterocycloalkyl, substituted or In unsubstituted C ₃ -C ₁₂ amide, the substituent is selected from hydroxyl and carbonyl.

In some embodiments, the linkage in the targeting group is preferably from the following fragments: , , , , , and , where each m is independently an integer from 1 to 6, each n, o, p is independently 0 or 1, and each q ¹ and q ² are independently 0, 1 or 2.

In some embodiments, the linkage in the targeting group is more preferably from the following fragments: , , , , , .

In some embodiments, the targeting moiety in the targeting group has the following structural fragment, n'' is independently 1 or 2 respectively.

In some embodiments, the targeting group has the following structure: GLO-1 GLS-1 GLO-2 GLS-2 GLO-3 GLS-3 GLO-4 GLS-4 GLO-5 GLS-5 GLO-6 GLS-6 GLO-7 GLS-7 GLO-8 GLS-8 GLO-9 GLS-9 GLO-10 GLS-10 GLO-11 GLS-11 GLO-12 GLS-12 GLO-13 GLS-13 GLO-14 GLS-14 GLO-15 GLS-15 GLO-16 GLS-16.

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.

In some embodiments, the antisense strand contains an inverted abasic residue at the 3'-end. In some embodiments, the sense strand contains one or two reverse abasic residues at the 3' or/and 5' end.

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

In some embodiments, at least one strand includes a 3' overhang that is at least 1 nucleotide long. In some embodiments, at least one strand includes a 3' overhang that is at least 2 nucleotides long. In one embodiment, the sense strand has a 1-10 nucleotide overhang at the 3'-end and/or 5'-end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or a 10-nucleotide overhang.

Another aspect of the invention provides a double-stranded ribonucleic acid (dsRNA) reagent expressing complement component C3, the dsRNA reagent comprising a sense strand and an antisense strand, wherein the length of each strand is 14 to 30 nucleotides, Comprises a region complementary to the C3 RNA transcript at nucleotide positions 2 to 18 in the antisense strand, wherein the complementary region contains a 0, 1, 2, or 3 nucleotide difference from one of the antisense sequences listed in Formula I At least 15 consecutive nucleotides of nucleotides, and optionally includes a targeting ligand: Double-stranded ribonucleic acid (dsRNA) reagent Antisense strand: 5'-N _a -(A ₁ A ₂ A ₃ A ₄ ) iN _b –(B ₁ B ₂ B ₃ B ₄ ) -N _b -(C ₁ C ₂ C ₃ C ₄ C ₅ )j -N _a - 3' where: i and j are independently selected from 0 or 1; each Each N _a and N _b independently represent 0-17 oligonucleotides; A ₁ A ₂ A ₃ A ₄ represents four consecutive nucleotides, which are represented in turn by lowercase 2'-OMe, uppercase 2'-fluoro, lowercase 2'-OMe, a motif modified by lowercase 2'-OMe, preferably between A ₁ and A ₂ , and between A ₂ and A ₃ , further including a phosphorothioate internucleoside linkage; B ₁ B ₂ B ₃ B ₄ represents a motif in which four consecutive nucleotides are all modified with lowercase 2'-OMe; C ₁ C ₂ C ₃ C ₄ C ₅ represents five consecutive nucleotides are all modified with lowercase 2'-OMe One of the modified motifs, preferably between _C3 and _C4 , and between _C4 and _C5 , further includes a phosphorothioate internucleoside linkage.

In some embodiments, _Na each independently represents 0 oligonucleotides, N _b each independently represents 2-5 oligonucleotides chemically modified as used herein, and i and j respectively represent 1.

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

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

In some embodiments, the sense strand contains one or two reverse abasic residues at the 3' or/and 5' end.

In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand includes a 3' overhang that is at least 1 nucleotide long. In some embodiments, at least one strand includes a 3' overhang that is at least 2 nucleotides long. In one embodiment, the sense strand has a 1-10 nucleotide overhang at the 3'-end and/or 5'-end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang.

In some embodiments, a dsRNA agent contains at least one modified nucleotide and further contains one or more targeting 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 includes N-acetyl-galactosamine (GalNAc). In some embodiments, the targeting group is as used herein, 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. Any one of GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16.

According to another aspect of the invention, there is provided a composition comprising any embodiment of the above-described dsRNA agent aspect of the invention.

In certain embodiments, the composition further includes a pharmaceutically acceptable carrier. In some embodiments, the composition further includes one or more additional therapeutic agents.

In certain embodiments, the compositions are packaged in a kit, container, wrapper, dispenser, prefilled syringe, or vial.

In some embodiments, the compositions are formulated for subcutaneous administration or are formulated for intravenous (IV) administration.

According to another aspect of the invention, there is provided a cell comprising any embodiment of the above-described dsRNA agent aspect of the invention.

In some embodiments, the cells are mammalian cells, optionally human cells.

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

In certain embodiments, the method further includes: (ii) maintaining the prepared cells for a sufficient time to obtain degradation of the mRNA transcript of the C3 gene, thereby inhibiting the expression of the C3 gene in the cell.

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

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

In certain embodiments, the method further includes assessing inhibition of the C3 gene after administering the dsRNA agent to the subject, wherein the assessment includes: (i) determining one or more physiological characteristics of the C3-related disease or disorder in the subject , and (ii) comparing the determined physiological characteristic 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 is indicative of inhibition of C3 gene expression in the subject exists or does not exist.

In some embodiments, the physiological characteristic determined is C3 levels in the blood;

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

According to another aspect of the present invention, there is provided a method of inhibiting C3 gene expression in a subject, which includes administering to the subject an effective amount of an embodiment of the foregoing dsRNA agent or an embodiment of the foregoing composition.

In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In certain embodiments, the dsRNA agent is administered to the subject via IV administration. In some embodiments, the method further includes: assessing suppression of the C3 gene after administration of the dsRNA agent, wherein the assessing means includes: (i) determining one or more physiological characteristics of the C3-related disease or condition in the subject; (ii) The determined physiological characteristic is compared 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 is indicative of the presence or absence of inhibition of C3 gene expression in the subject. In some embodiments, the physiological characteristic determined is C3 levels in the blood; in some embodiments, paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disease involving a complement-mediated vascular Endohemolysis is a physiological feature defined by acquired hemolytic anemia, hemoglobinuria, bone marrow failure, and thrombophilia (tendency to form blood clots). A decrease in C3 levels and/or C3 in the blood indicates a decrease in C3 gene expression in the subject.

According to another aspect of the present invention, there is provided a method of treating a disease or disorder associated with C3 protein, which includes: administering to a subject an effective amount of any embodiment of the foregoing dsRNA agent aspect of the present invention or the foregoing composition of the present invention. Any embodiment to inhibit C3 gene expression.

In certain embodiments, the C3-associated disorder is selected from: kidney-associated 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 other organ C3-related Diseases 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), antineutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegeneration Diseases, preferably C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), lupus nephritis, IgA nephropathy (IgAN) and primary membranous nephropathy Kidney disease, age-related macular degeneration (AMD).

In some embodiments, the method further includes administering an additional treatment regimen to the subject.

In some embodiments, additional treatment options include treatment of C3-related diseases or conditions. In certain embodiments, additional treatment regimens include: administering to the subject one or more C3 antisense polynucleotides of the invention; administering to the subject a non-C3 dsRNA therapeutic; and effecting behavioral changes in the subject. In some embodiments, the non-C3 dsRNA therapeutic is one of: 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 and/or agents that are C3 peptide inhibitors (e.g., simvastatin) to treat subjects with diseases that would benefit from C3 reduction.

In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In certain embodiments, the dsRNA agent is administered to the subject via IV administration. In some embodiments, the method further includes determining the efficacy of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject. In some embodiments, means for determining the efficacy of a treatment in a subject includes: (i) determining one or more physiological characteristics of a C3-related disease or disorder in the subject; (ii) correlating the determined physiological characteristics with the C3-related disease or disorder Baseline pre-treatment physiological characteristics of the condition are compared, wherein the comparison indicates one or more of the presence, absence, and level of efficacy of a double-stranded ribonucleic acid (dsRNA) agent administered to the subject. In some embodiments, the physiological characteristic determined is C3 levels in the blood; in some embodiments, paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disease involving a complement-mediated vascular Endohemolysis is a physiological feature defined by acquired hemolytic anemia, 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.

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

According to another aspect of the invention, there is provided a method of altering the physiological characteristics of a C3-related disease or disorder in a subject as compared to the baseline pre-treatment physiological characteristics of the C3-related disease or disorder in the subject, the method comprising administering to the subject an effective amount of the present invention. Any embodiment of the foregoing dsRNA agent aspect or any embodiment of the foregoing composition of the invention is invented to alter the physiological characteristics of a C3-related disease or disorder in a 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 reduction in hemolysis. Sequence description

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

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

SEQ ID NO: 1 is human C3 mRNA [NCBI reference sequence: NM_000064.4].

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

In the sequences shown in Table 3, the delivery molecules used in the in vivo studies are designated "GLX-n" at the 3' or 5' end of each sense strand. Chemical modifications are represented as: uppercase: 2'-Fluoro; lowercase: 2'-OMe; phosphorothioate: *; UNA: open ring nucleic acid; Invab = reverse abasic.

Some embodiments of the invention include RNAi agents capable of inhibiting expression of the complement component C3 gene, such as, but not limited to, double-stranded (ds) RNAi agents. The C3 dsRNA reagents of the present invention can target C3 transcripts, resulting in the inhibition of C3 protein expression. Some embodiments of the invention also include compositions comprising C3 RNAi agents and methods of using the compositions. The C3 RNAi agents disclosed herein can be attached to delivery compounds for delivery to cells, including delivery to hepatocytes. Pharmaceutical compositions of the present invention may comprise at least one C3 dsRNA agent and a delivery compound. In some embodiments of the invention, the delivery compound is a GalNAc-containing delivery compound. C3 RNAi agents delivered to cells inhibit C3 gene expression, thereby reducing the activity of the gene's C3 protein product in the cell. The dsRNAi agents of the invention can be used to treat C3-related diseases and disorders. 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.

In some embodiments of the invention, reducing C3 expression in a cell or subject treats a disease or disorder associated with C3 expression in a cell or subject, respectively. Non-limiting examples of diseases and conditions that may be treated by reducing C3 activity are: kidney-related C3 glomerulopathy (C3G), immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious disease Glomerulonephritis (PIGN), systemic lupus erythematosus, lupus nephritis, ischemia/reperfusion injury and IgA nephropathy, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and other organs C3-related diseases such as neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), primary membranous nephropathy, and complement dysfunction in other organs are also known to be associated with Diseases such as age-related macular degeneration (AMD), rheumatoid arthritis (RA), antineutrophil 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 (IgA N) and Primary membranous nephropathy, age-related macular degeneration (AMD).

Described below are how to prepare and use compositions containing C3 single-stranded (ssRNA) and double-stranded (dsRNA) agents to inhibit C3 gene expression, as well as compositions and methods for treating diseases and conditions caused or modulated by C3 gene expression. . As used herein, diseases and/or conditions caused or modulated by the presence and/or levels of C3 gene expression are referred to as "C3-associated diseases and/or conditions." The term "RNAi" is also known in the art and may be referred to as "siRNA".

As used herein, the term "RNAi" refers to agents that contain RNA and mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. As is known in the art, an RNAi target region refers to a contiguous portion of the nucleotide sequence of an RNA molecule formed during gene transcription, which includes messenger RNA (mRNA), which is a processing product of the primary transcript RNA. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion. The target sequence can be 8-30 nucleotides long (inclusive), 10-30 nucleotides long (inclusive), 12-25 nucleotides long (inclusive), 15-23 nucleotides long (inclusive), 16 - 23 nucleotides long (inclusive), or 18 - 23 nucleotides long (inclusive), including all shorter within each specified range length. In some embodiments of the invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleosides Sour and 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 invention, the target sequences are 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 long, the sequence is completely or at least substantially complementary to at least a portion of the RNA transcript of the C3 gene.

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, C3 RNAi as described herein inhibits expression of C3 protein.

In some embodiments of the invention, pharmaceutical compositions include one, two, three, or more independent anti-C3 dsRNA agents, and may also include one or more independently selected delivery compounds. In some embodiments, two, three, four or more anti-C3 dsRNAs capable of targeting one, two, three, four or more different positions/regions of C3 mRNA are administered to in a cell or object.

As used herein, a "dsRNA agent" refers to a composition comprising an RNA or RNA-like (eg, chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of a target mRNA transcript. While not wishing to be bound to a particular theory, the dsRNA agents of the present invention may act through an RNA interference mechanism (i.e., induce RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC) ), or act through any alternative mechanism or pathway. Methods to achieve 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 which are incorporated herein by reference in their entirety. Gene silencing means known in the art can be used in conjunction with the disclosure provided herein to achieve inhibition of C3 expression.

The dsRNA reagent disclosed herein consists of a sense strand and an antisense strand, including but not limited to: short interfering RNA (siRNA), RNAi reagents, microRNA (miRNA), short hairpin RNA (shRNA) and Dicer. substrate. The antisense strand of the dsRNA reagents described herein is at least partially complementary to the targeted mRNA, and it is understood in the art that dsRNA duplex structures of various lengths can be used to inhibit target gene expression. For example, dsRNAs with duplex structures of 19, 20, 21, 22, and 23 base pairs are known to effectively induce RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). It is also known in the art that shorter or longer RNA duplex structures are also effective in inducing RNA interference. The C3 dsRNA in certain embodiments of the 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 shorter length. It can also be effective to reduce 4 nucleotides at one or both ends compared to the dsRNA listed in Tables 1-3 respectively. In some embodiments of the invention, the C3 dsRNA agent may have a partial sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one or more sequences of Tables 1-3 , and their ability to inhibit C3 gene expression does not differ by more than 5%, 10%, 15%, 20%, 25%, or 30%. The sense sequences, antisense sequences and duplexes disclosed in Tables 1-3 may be referred to as "parent" sequences herein, which means that the sequences disclosed in Tables 1-3 may be modified, shortened, extended, include substitutions, etc. , the resulting sequence retains all or at least part of the efficacy of its parent sequence in the methods and compositions of the invention, as described herein.

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

Table 1 includes the sense and antisense strands and provides the identification number of the duplex formed by the sense and antisense strands on the same row in Table 1. In certain embodiments of the invention, the antisense sequence contains nucleobase u or nucleobase a in position 1 thereof. In certain embodiments of the 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 a position in each strand that "pairs" with each other when the two strands act 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, nucleobase position 2 of the sense strand is in a matching position with position 22 of the antisense strand. In another non-limiting example, in the 18-nucleobase sense strand and the 18-nucleobase antisense strand, the nucleobase at position 1 of the sense strand is in a matching position with the nucleobase at position 18 of the antisense strand; and the sense strand Nucleobase 4 in the strand matches nucleobase 15 in the antisense strand. The skilled person will understand how to identify matching positions between the sense and antisense strands of duplexes and paired strands.

The first column in Table 1 represents the duplex AV# of the duplex containing the 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 invention, each containing the sense and antisense sequences shown in the same row, and the assigned identifier for each duplex is shown at the end of that row. in one column.

In some embodiments of the methods of the invention, an RNAi agent comprising the polynucleotide sequence set forth in Table 1 is administered to the subject. In some embodiments of the invention, the RNAi agent administered to the subject includes 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 method of the present invention, it is also included in linking an RNAi agent of the polynucleotide sequence shown in Table 1 to a delivery molecule, a non-limiting example of which is a delivery compound comprising GalNAc.

Table 1: Unmodified antisense and sense strand sequences of C3 RNAi reagents. All sequences are shown in 5' to 3' orientation. Duplex AV# is the number assigned to the duplex of both strands in the same row in 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 sense sequence shown in the fourth column. The sequence number of the antisense sequence is shown. Duplex AV# The base sequence of the sense strand is 5'→3' SEQ ID NO Antisense strand base sequence 5' → 3' SEQ ID NO AV00375 CUAUCGGAUCUUCACCGUCAA 2 UUGACGGUGAAGAUCCGAUAG 75 AV00376 GAUCCGAGCCUACUAUGAAAA 3 UUUUCAUAGUAGGCUCGGAUC 76 AV00377 GAGAAAUUCUACUACAUCUAA 4 UUAGAUGUAGUAGAAUUUCUC 77 AV00378 GAAUUCUACUACAUCUAUAAA 5 UUUUAAGAUGUAGUAGAAUUC 78 AV00379 GGCCUUUGUCAUCUUCGGGAA 6 UUCCCGAAGAUGACAAAGGCC 79 AV00380 GAUCCCCAUCGUGACCUCA 7 UGAGAGGUCACGAUGGGGAUC 80 AV00381 CAAACCAGGAAUGCCCUUUGA 8 UCAAAGGGCAUUCCUGGUUUG 81 AV00382 GGAAUGCCCUUUGACCUCAUA 9 UAUGAGGUCAAAGGGCAUUCC 82 AV00383 ACUCCAACAAUUACCUGCAUA 10 UAUGCAGGUAAUUGUUGGAGU 83 AV00384 CGUCAACUUCCUCCUGCGAAA 11 UUUCGCAGGAGGAAGUUGACG 84 AV00385 CGCUACUACACCUACCUGAUA 12 UAUCAGGUAGGUGUAGUAGCG 85 AV00386 GUUCGUGCUGAAUAAGAAGAA 13 UUCUUCUUAUUCAGCACGAAC 86 AV00387 GGAGAACCCCAUGAGGUUCUA 14 UAGAACCUCAUGGGGUUCCC 87 AV00388 GUUGCAGAAGAGAACAUCGUA 15 UACGAUGUUCUCUUCUGCAAC 88 AV00389 GGAGAACAUCGUUUCCCGAAA 16 UUUCGGGAAACGAUGUUCUCC 89 AV00390 AGAACAUCGUUUCCCGAAGUA 17 UACUUCGGGAAACGAUGUUCU 90 AV00391 GUCACAGUAAUGCAGGACUUA 18 UAAGUCCUGCAUUACUGUGAC 91 AV00392 ACAGUAAUGCAGGACUUCUUA 19 UAAGAAGUCCUGCAUUACUGU 92 AV00393 ACCCUACUCUGUUGUUCGAAA 20 UUUCGAACAACAGAGUAGGGU 93 AV00394 CCCUACUCUGUUGUUCGAAAA twenty one UUUUCGAACAACAGAGUAGGG 94 AV00395 CUACUCUGUUGUUCGAAACGA twenty two UCGUUUCGAACAACAGAGUAG 95 AV00396 CUACUCCACAAUCCAGCCUUA twenty three UAAGGCUGGAUUGUGGAGUAG 96 AV00397 CAUUUCAUCAGUGACGGUGUA twenty four UACACCGUCACUGAUGAAAUG 97 AV00398 GGUCAUCGCUGUGCAUUACCA 25 UGGUAAUGCACAGCGAUGACC 98 AV00399 CUGUGCAUUACCUGGAUGAAA 26 UUUCAUCCAGGUAAUGCACAG 99 AV00400 GUGCAUUACCUGGAUGAAACA 27 UGUUUCAUCCAGGUAAUGCAC 100 AV00401 GUCUCUCUGGCUGUCAACCUA 28 UAGGUUGACAGCCAGAGAGAC 101 AV00402 GGAGACUUCCUUGAAGCCAAA 29 UUUGGCUUCAAGGAAGUCUCC 102 AV00403 GACUUCCUUGAAGCCAACUAA 30 UUAGUUGGCUUCAAGGAAGUC 103 AV00404 CUUGAAGCCAACUACAUGAAA 31 UUUCAUGUAGUUGGCUUCAAG 104 AV00405 AGCCAACUACAUGAACCUACA 32 UGUAGGUUCAUGUAGUUGGCU 105 AV00406 CUUAACAAAUUUCUGACCACA 33 UGUGGUCAGAAAUUUGUUAAG 106 AV00407 GACCACAGCCAAAGAUAAGAA 34 UUCUUAUCUUUGGCUGUGGUC 107 AV00408 CCUACUGCAGCUAAAAGACUA 35 UAGUCUUUUAGCUGCAGUAGG 108 AV00409 CUGCAGCUAAAGACUUUGAA 36 UUCAAAGUCUUUUAGCUGCAG 109 AV00410 UGCAGCUAAAAGACUUUGACA 37 UGUCAAAGUCUUUUAGCUGCA 110 AV00411 GCAGCUAAAAGACUUUGACUA 38 UAGUCAAAGUCUUUUAGCUGC 111 AV00412 GCUCAAUGAACAGAGAUAACUA 39 UAGUAUCUCUGUUCAUUGAGC 112 AV00413 CUCAAUGAACAGAGAUACUAA 40 UUAGUAUCUCUGUUCAUUGAG 113 AV00414 CAAUGAACAGAGAUACUACGA 41 UCGUAGUAUCUCUGUUCAUUG 114 AV00415 UGUUCCAAGCCUUGGCUCAAA 42 UUUGAGCCAAGGCUUGGAACA 115 AV00416 GUUCCAAGCCUUGGCUCAAUA 43 UAUUGAGCCAAGGCUUGGAAC 116 AV00417 CCUUGGCUCAAUACCAAAAGA 44 UCUUUUGGUAUUGAGCCAAGG 117 AV00418 GAGGAAAAUGAGGGUUUCACA 45 UGUGAAACCCUCAUUUUCCUC 118 AV00419 GGUGGUGACAAUGUACCAUGA 46 UCAUGGUACAUUGUCACCACC 119 AV00420 UGGUGACAAUGUACCAUGCUA 47 UAGCAUGGUACAUUGUCACCA 120 AV00421 AUCAACUCACCUGUAAUAAA 48 UUUUAUUACAGGUGAGUUGAU 121 AV00422 CUCACCUGUAAUAAAUUCGAA 49 UUCGAAUUUAUUACAGGUGAG 122 AV00423 UCACCUGUAAUAAAUUCGACA 50 UGUCGAAUUUAUUACAGGUGA 123 AV00424 CCUCAAGGUCACCAUAAAACA 51 UGUUUUAUGGACCUUGAGG 124 AV00425 UGCCAAGAACACUAUGAUCCA 52 UGGAUCAUAGUGUUCUUGGCA 125 AV00426 GGAACACUAUGAUCCUUGAGA 53 UCUCAAGGAUCAUAGUGUUCC 126 AV00427 UAUGAUCCUUGAGAUCUGUAA 54 UUACAGAUCUCAAGGAUCAUA 127 AV00428 CUUGAGAUCUGUACCAGGUAA 55 UUACCUGGUACAGAUCUCAAG 128 AV00429 GGAGACCAGGAUGCCACUAUA 56 UAUAGUGGCAUCCUGGUCUCC 129 AV00430 AUUGGACAUAUCCAUGAUGAA 57 UUCAUCAUGGAUAUGUCCAAU 130 AV00431 GUGGACAUAUCCAUGAUGACA 58 UGUCAUCAUGGAUAUGUCCAC 131 AV00432 ACAGAUACAUCUCCAAGUAUA 59 UAUACUUGGAGAUGUAUCUGU 132 AV00433 CAAGUAUGAGCUGGACAAAGA 60 UCUUUGUCCAGCUCAUACUUG 133 AV00434 AGGUCUCACACUCUGAGGAUA 61 UAUCCUCAGAGUGUGAGACCCU 134 AV00435 GUCUAGCUUUCAAAGUUCACA 62 UGUGAACUUUGAAAGCUAGAC 135 AV00436 GCUUUAAUGUAGAGCUUAUCA 63 UGAUAAGCUCUACAUUAAAGC 136 AV00437 GUCUACGCCUAUUACAACCUA 64 UAGGUUGUAAUAGGCGUAGAC 137 AV00438 GGAAUUGCUUCAUACAAAAGA 65 UCUUUUGUAUGAAGCAAUUCC 138 AV00439 CAGGAGUGGACUAUGUGUACA 66 UGUACACAAUAGUCCACUCCUG 139 AV00440 CAGCUGUCCAAUGACUUUGAA 67 UUCAAAGUCAUUGGACAGCUG 140 AV00441 CUGUCCAAUGACUUUGACGAA 68 UUCGUCAAAGUCAUUGGACAG 141 AV00442 GGUCCAAUGACUUUGACGAGA 69 UCUCGUCAAAGUCAUUGGACC 142 AV00443 UCCAAUGACUUUGACGAGUAA 70 UUACUCGUCAAAGUCAUUGGA 143 AV00444 GCUUUGACGAGUACAUCAUGA 71 UCAUGAUGUACUCGUCAAAGC 144 AV00445 GAGUACAUCAUGGCCAUUGAA 72 UUCAAUGGCCAUGAUGUACUC 145 AV00446 AGAAGAAACACUACCUCAUGA 73 UCAUGAGGUAGUGUUUCUUCU 146 AV00447 GAAGAAACACUACCUCAUGUA 74 UACAUGAGGUAGUGUUUCUUC 147

Table 2 shows the antisense and sense strand sequences of certain chemically modified C3 RNAi agents of the invention. In some embodiments of the methods of the invention, an RNAi agent having the polynucleotide sequence set forth in Table 2 is administered to the cell and/or subject. In some embodiments of the methods of the invention, an RNAi agent having the polynucleotide sequence set forth in Table 2 is administered to the subject. In some embodiments of the invention, the RNAi agent administered to the subject comprises the duplexes noted in the first column of Table 2, and comprises the sense and reverse duplexes shown in the third and sixth columns of the same row in Table 2, respectively. Sequence modifications in the sense strand sequence. In some embodiments of the methods of the invention, the sequences shown in Table 2 can be linked to (also referred to herein as "conjugated to") a compound capable of delivering an RNAi agent to cells and/or tissues of a subject. Non-limiting examples of delivery compounds useful in certain embodiments of the invention are GalNAc-containing compounds. In Table 2, the first column represents the duplex AV# of the base sequence and corresponds to Table 1. For the base sequence identified by duplex AV#, not only the base sequences contained in the sense and antisense strands are shown, but also with the specified chemical modifications shown in the same row of Table 2. For example, the first row of Table 1 shows the single-stranded sequences of sense and antisense bases, which together form a duplex, identified as: duplex AV# AV00375; and in the duplex AV# AV00375 listed in Table 2, As a duplex, it contains the base sequences of AV00375-SS and AV00375-AS, and contains 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 for the sense sequence (including modifications) shown in the third column of the same row. "Antisense Strand AS#" in the fifth column of Table 2 is the designated identifier for 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. "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.

Table 2: Provides chemically modified C3 RNAi reagent antisense and sense strand sequences. All sequences are shown 5' to 3'. These sequences were used in certain in vitro testing studies described herein. Chemical modifications are expressed as: uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *; UNA: open ring nucleic acid. Duplex AV# Chain of Justice SS# justice sequence SEQ ID NO Antisense strand AS# antisense sequence SEQ ID NO AV00375 AV00375-SS c*u*aucggaUcUuCaccguca*a 148 AV00375-AS u*U*gaCg(gUNA)ugaaGaUcCgau*a*g 221 AV00376 AV00376-SS g*a*uccgagCcUaCuaugaaa*a 149 AV00376-AS u*U*uucaUaguaGgCuCgga*u*c 222 AV00377 AV00377-SS g*a*gaaauuCuAcUacaucua*a 150 AV00377-AS u*U*agauGuaguAgAaUuuc*u*c 223 AV00378 AV00378-SS g*a*auucuaCuAcAucuauaa*a 151 AV00378-AS u*U*uauaGauguAgUaGaau*u*c 224 AV00379 AV00379-SS g*g*ccuuugUcAuCuucggga*a 152 AV00379-AS u*U*ccCg(aUNA)agauGaCaAagg*c*c 225 AV00380 AV00380-SS g*a*uccccaUcGuGaccucuc*a 153 AV00380-AS u*G*agAg(gUNA)ucacGaUgGgga*u*c 226 AV00381 AV00381-SS c*a*aaccagGaAuGcccuuug*a 154 AV00381-AS u*C*aaAg(gUNA)gcauUcCuGguu*u*g 227 AV00382 AV00382-SS g*g*aaugccCuUuGaccucau*a 155 AV00382-AS u*A*ugagGucaaAgGgCauu*c*c 228 AV00383 AV00383-SS a*c*uccaacAaUuAccugcau*a 156 AV00383-AS u*A*ugCa(gUNA)guaaUuGuUgga*g*u 229 AV00384 AV00384-SS c*g*ucaacuUcCuCcugcgaa*a 157 AV00384-AS u*U*ucGc(aUNA)ggagGaAgUuga*c*g 230 AV00385 AV00385-SS c*g*cuacuaCaCcUaccugau*a 158 AV00385-AS u*A*ucagGuaggUgUaGuag*c*g 231 AV00386 AV00386-SS g*u*ucgugcUgAaUaagaaga*a 159 AV00386-AS u*U*cuucUuauuCaGcAcga*a*c 232 AV00387 AV00387-SS g*g*agaaccCcAuGagguucu*a 160 AV00387-AS u*A*gaacCucauGgGgUucu*c*c 233 AV00388 AV00388-SS g*u*ugcagaAgAgAacaucgu*a 161 AV00388-AS u*A*cgauGuucuCuUcUgca*a*c 234 AV00389 AV00389-SS g*g*agaacaUcGuUucccgaa*a 162 AV00389-AS u*U*ucGg(gUNA)aaacGaUgUucu*c*c 235 AV00390 AV00390-SS a*g*aacaucGuUuCccgaagu*a 163 AV00390-AS u*A*cuUc(gUNA)ggaaAcGaUguu*c*u 236 AV00391 AV00391-SS g*u*cacaguAaUgCaggacuu*a 164 AV00391-AS u*A*agucCugcaUuAcUgug*a*c 237 AV00392 AV00392-SS a*c*aguaauGcAgGacuucuu*a 165 AV00392-AS u*A*agaaGuccuGcAuUacu*g*u 238 AV00393 AV00393-SS a*c*ccuacuCuGuUguucgaa*a 166 AV00393-AS u*U*ucgaAcaacAgAgUagg*g*u 239 AV00394 AV00394-SS c*c*cuacucUgUuGuucgaaa*a 167 AV00394-AS u*U*uucgAacaaCaGaGuag*g*g 240 AV00395 AV00395-SS c*u*acucugUuGuUcgaaacg*a 168 AV00395-AS u*C*guUu(cUNA)gaacAaCaGagu*a*g 241 AV00396 AV00396-SS c*u*acuccaCaAuCcagccuu*a 169 AV00396-AS u*A*agGc(uUNA)ggauUgUgGagu*a*g 242 AV00397 AV00397-SS c*a*uuucauCaGuGacggugu*a 170 AV00397-AS u*A*caCc(gUNA)ucacUgAuGaaa*u*g 243 AV00398 AV00398-SS g*g*ucaucgCuGuGcauuacc*a 171 AV00398-AS u*G*guaaUgcacAgCgAuga*c*c 244 AV00399 AV00399-SS c*u*gugcauUaCcUggaugaa*a 172 AV00399-AS u*U*ucauCcaggUaAuGcac*a*g 245 AV00400 AV00400-SS g*u*gcauuaCcUgGaugaaac*a 173 AV00400-AS u*G*uuucAuccaGgUaAugc*a*c 246 AV00401 AV00401-SS g*u*cucucuGgCuGucaaccu*a 174 AV00401-AS u*A*gguuGacagCcAgAgag*a*c 247 AV00402 AV00402-SS g*g*agacuuCcUuGaagccaa*a 175 AV00402-AS u*U*uggcUucaaGgAaGucu*c*c 248 AV00403 AV00403-SS g*a*cuuccuUgAaGccaacua*a 176 AV00403-AS u*U*aguuGgcuuCaAgGaag*u*c 249 AV00404 AV00404-SS c*u*ugaagcCaAcUacaugaa*a 177 AV00404-AS u*U*ucauGuaguUgGcUuca*a*g 250 AV00405 AV00405-SS a*g*ccaacuAcAuGaaccuac*a 178 AV00405-AS u*G*uaggUucauGuAgUugg*c*u 251 AV00406 AV00406-SS c*u*uaacaaAuUuCugacca*a 179 AV00406-AS u*G*ugGu(cUNA)agaaAuUuGuua*a*g 252 AV00407 AV00407-SS g*a*ccacagCcAaAgauaaga*a 180 AV00407-AS u*U*cuuaUcuuuGgCuGugg*u*c 253 AV00408 AV00408-SS c*c*uacugcAgCuAaaagacu*a 181 AV00408-AS u*A*gucuUuuagCuGcAgua*g*g 254 AV00409 AV00409-SS c*u*gcagcuAaAaGacuuuga*a 182 AV00409-AS u*U*caaaGucuuUuAgCugc*a*g 255 AV00410 AV00410-SS u*g*cagcuaAaAgAcuuugac*a 183 AV00410-AS u*G*ucaaAgucuUuUaGcug*c*a 256 AV00411 AV00411-SS g*c*agcuaaAaGaCuuugacu*a 184 AV00411-AS u*A*gucaAagucUuUuAgcu*g*c 257 AV00412 AV00412-SS g*c*ucaaugAaCaGagauacu*a 185 AV00412-AS u*A*guauCucugUuCaUuga*g*c 258 AV00413 AV00413-SS c*u*caaugaAcAgAgauacua*a 186 AV00413-AS u*U*aguaUcucuGuUcAuug*a*g 259 AV00414 AV00414-SS c*a*augaacAgAgAuacuacg*a 187 AV00414-AS u*C*guagUaucuCuGuUcau*u*g 260 AV00415 AV00415-SS u*g*uuccaaGcCuUggcucaa*a 188 AV00415-AS u*U*ugAg(cUNA)caagGcUuGgaa*c*a 261 AV00416 AV00416-SS g*u*uccaagCcUuGgcucaau*a 189 AV00416-AS u*A*uugaGccaaGgCuUgga*a*c 262 AV00417 AV00417-SS c*c*uuggcuCaAuAccaaaag*a 190 AV00417-AS u*C*uuuuGguauUgAgCcaa*g*g 263 AV00418 AV00418-SS g*a*ggaaaaUgAgGguuucac*a 191 AV00418-AS u*G*ugaaAcccuCaUuUucc*u*c 264 AV00419 AV00419-SS g*g*uggugaCaAuGuaccaug*a 192 AV00419-AS u*C*auggUacauUgUcAcca*c*c 265 AV00420 AV00420-SS u*g*gugacaAuGuAccaugcu*a 193 AV00420-AS u*A*gcAu(gUNA)guacAuUgUcac*c*a 266 AV00421 AV00421-SS a*u*caacucAcCuGuaauaaa*a 194 AV00421-AS u*U*uuauUacagGuGaGuug*a*u 267 AV00422 AV00422-SS c*u*caccugUaAuAaauucga*a 195 AV00422-AS u*U*cgaaUuuauUaCaGgug*a*g 268 AV00423 AV00423-SS u*c*accuguAaUaAauucgac*a 196 AV00423-AS u*G*ucgaAuuuaUuAcAggu*g*a 269 AV00424 AV00424-SS c*c*ucaaggUcAcCauaaaac*a 197 AV00424-AS u*G*uuuuAugguGaCcUuga*g*g 270 AV00425 AV00425-SS u*g*ccaagaAcAcUaugaucc*a 198 AV00425-AS u*G*gaucAuaguGuUcUugg*c*a 271 AV00426 AV00426-SS g*g*aacacuAuGaUccuugag*a 199 AV00426-AS u*C*ucAa(gUNA)gaucAuAgUguu*c*c 272 AV00427 AV00427-SS u*a*ugauccUuGaGaucugua*a 200 AV00427-AS u*U*acagAucucAaGgAuca*u*a 273 AV00428 AV00428-SS c*u*ugagauCuGuAccaggua*a 201 AV00428-AS u*U*acCu(gUNA)guacAgAuCuca*a*g 274 AV00429 AV00429-SS g*g*agaccaGgAuGccacuau*a 202 AV00429-AS u*A*uaguGgcauCcUgGucu*c*c 275 AV00430 AV00430-SS a*u*uggacaUaUcCaugauga*a 203 AV00430-AS u*U*caucAuggaUaUgUcca*a*u 276 AV00431 AV00431-SS g*u*ggacauAuCcAugaugac*a 204 AV00431-AS u*G*ucauCauggAuAuGucc*a*c 277 AV00432 AV00432-SS a*c*agauacAuCuCcaaguau*a 205 AV00432-AS u*A*uacuUggagAuGuAucu*g*u 278 AV00433 AV00433-SS c*a*aguaugAgCuGgacaaag*a 206 AV00433-AS u*C*uuugUccagCuCaUacu*u*g 279 AV00434 AV00434-SS a*g*gucucaCaCuCugaggau*a 207 AV00434-AS u*A*uccuCagagUgUgAgac*c*u 280 AV00435 AV00435-SS g*u*cuagcuUuCaAaguucac*a 208 AV00435-AS u*G*ugaaCuuugAaAgCuag*a*c 281 AV00436 AV00436-SS g*c*uuuaauGuAgAgcuuauc*a 209 AV00436-AS u*G*auaaGcucuAcAuUaaa*g*c 282 AV00437 AV00437-SS g*u*cuacgcCuAuUacaaccu*a 210 AV00437-AS u*A*gguuGuaauAgGcGuag*a*c 283 AV00438 AV00438-SS g*g*aauugcUuCaUacaaaag*a 211 AV00438-AS u*C*uuuuGuaugAaGcAauu*c*c 284 AV00439 AV00439-SS c*a*ggagugGaCuAuguguac*a 212 AV00439-AS u*G*uacaCauagUcCaCucc*u*g 285 AV00440 AV00440-SS c*a*gcugucCaAuGacuuuga*a 213 AV00440-AS u*U*caaaGucauUgGaCagc*u*g 286 AV00441 AV00441-SS c*u*guccaaUgAcUuugacga*a 214 AV00441-AS u*U*cgucAaaguCaUuGgac*a*g 287 AV00442 AV00442-SS g*g*uccaauGaCuUugacgag*a 215 AV00442-AS u*C*ucGu(cUNA)aaagUcAuUgga*c*c 288 AV00443 AV00443-SS u*c*caaugaCuUuGacgagua*a 216 AV00443-AS u*U*acucGucaaAgUcAuug*g*a 289 AV00444 AV00444-SS g*c*uuugacGaGuAcaucaug*a 217 AV00444-AS u*C*augaUguacUcGuCaaa*g*c 290 AV00445 AV00445-SS g*a*guacauCaUgGccauuga*a 218 AV00445-AS u*U*caauGgccaUgAuGuac*u*c 291 AV00446 AV00446-SS a*g*aagaaaCaCuAccucaug*a 219 AV00446-AS u*C*auGa(gUNA)guagUgUuUcuu*c*u 292 AV00447 AV00447-SS g*a*agaaacAcUaCcucaugu*a 220 AV00447-AS u*A*caugAgguaGuGuUucu*u*c 293

Table 3 shows the antisense and sense strand sequences of certain chemically modified C3 RNAi agents of the invention. In some embodiments of the methods of the invention, an RNAi agent shown in Table 3 is administered to the cell and/or subject. In some embodiments of the methods of the invention, an RNAi agent having the polynucleotide sequence set forth in Table 3 is administered to the subject. In some embodiments of the invention, the RNAi agent administered to the subject comprises a duplex identified in the first column of Table 3, and comprises the sense and reverse duplexes, respectively, in the third and sixth columns of the same row of Table 3. The sequence shown in the sense strand sequence modifies and/or delivers the compound. This sequence was used in certain in vivo testing studies described elsewhere herein. In some embodiments of the methods of the invention, the sequences shown in Table 3 can be linked (also referred to herein as "conjugated") to a compound for delivery, a non-limiting example of which is a GalNAc-containing compound , that is, there is a delivery compound identified as “GLX-n” on the sense strand in the third column of Table 3. Certain embodiments of delivery compounds 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 GalNAc-containing compounds. As used herein, "GLX" is used to mean a "GLS" or "GLO" delivery compound ("X" can be "S" or "O"), and GLX-n can be any GLS and GLO that can be linked during synthesis Delivery compounds to the 3'- or 5'-end of the oligonucleotide. For example but not limited to this: GLX-13 and GLX-14 can be connected to the 3'-end of the oligonucleotide of the present invention during the synthesis process; GLX-5 and GLX-15 can be connected to the present invention during the synthesis process. the 5'-end of the oligonucleotide. In some embodiments, as used herein and as shown in Table 3, "GLX-n" is used to represent an attached GalNAc-containing compound, which is replaced with 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. Any one of GLO-14, GLO-15 and GLO-16. In some implementations, GLX-n is shown as an example but not limited to the ones disclosed in the prior art (Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958−16961) that can be used to connect GalNAc-containing compounds used in certain in vivo testing studies described elsewhere herein. One skilled in the art will be able to prepare and use the dsRNA compounds of the invention, wherein 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 structure of each of these is provided elsewhere in this article. The first column of Table 3 provides the duplex AD# assigned to the duplexes for the sense and antisense sequences in that row of the table. For example, duplex AD# AD00343 is a duplex composed of sense strand AD00343-SS and antisense strand AD00343-AS. Each row in Table 3 provides one sense strand and one antisense strand, and discloses the duplex consisting of the indicated sense strand and antisense strand. The "sense strand SS#" in the second column of Table 3 is the designated identifier for the sense sequence (including modifications) shown in the third column of the same row. "Antisense Strand AS#" in the fifth column of Table 3 is the designated identifier for the antisense sequence (including modifications) shown in the sixth column. The identifier for certain linked GalNAc-containing GLO compounds is shown as GLX-n, and it is understood that another of the GLO-n or GLS-n compounds may be substituted for the compound shown as GLX-n and the resulting compound is also included in the Compounds are included in embodiments of the methods and/or compositions of the invention. In Table 3, SEQ ID NO:294 to SEQ ID NO:342 and SEQ ID NO:392 to SEQ ID NO:406 are the sequences of the sense strand and SEQ ID NO:343 to SEQ ID NO:391 and SEQ ID NO:407 to SEQ ID NO:421 is the antisense strand sequence. In Table 3, the delivery molecules are represented as "GLS-5", "GLS-15" or "GLS-5" at the 3' or 5' end of the sense strand NO:294 to SEQ ID NO:342, NO:392 to SEQ ID NO:406 "GLX-n". Chemical modifications are represented as: uppercase: 2'-fluoro; lowercase: 2'-methoxy; phosphorothioate: *; Invab: reverse abasic.

Table 3 provides chemically modified C3 RNAi reagent antisense and sense strand sequences for in vivo testing. All sequences are shown 5' to 3'. These sequences were used in certain in vivo testing studies described elsewhere herein. The delivery molecules used in in vivo studies are designated "GLn-x" at the 5' or 3' end of each sense strand. Chemical modifications are expressed as: uppercase: 2'-fluoro; lowercase: 2'-OMe; phosphorothioate: *; Invab: "reverse abasic", UNA: open ring nucleic acid. Duplex AD# Chain of Justice SS# justice sequence SEQ ID NO Antisense strand AS# antisense sequence SEQ ID NO AD00343 AD00343-SS g*c*uccaacAaUuAccugcau*a(GLX-n) 294 AD00343-AS u*A*ugCa(gUNA)guaaUuGuUgga*g*c 343 AD00344 AD00344-SS c*g*agaacaUcGuUucccgaa*a(GLX-n) 295 AD00344-AS u*U*ucGg(gUNA)aaacGaUgUucu*c*g 344 AD00345 AD00345-SS g*c*uuggcuCaAuAccaaaag*a(GLX-n) 296 AD00345-AS u*C*uuuuGguauUgAgCcaa*g*c 345 AD00346 AD00346-SS c*g*uggugaCaAuGuaccaug*a(GLX-n) 297 AD00346-AS u*C*auggUacauUgUcAcca*c*g 346 AD00347 AD00347-SS g*g*gugacaAuGuAccaugcu*a(GLX-n) 298 AD00347-AS u*A*gcAu(gUNA)guacAuUgUcac*c*c 347 AD00348 AD00348-SS g*u*caacucAcCuGuaauaaa*a(GLX-n) 299 AD00348-AS u*U*uuauUacagGuGaGuug*a*c 348 AD00349 AD00349-SS g*c*accuguAaUaAauucgac*a(GLX-n) 300 AD00349-AS u*G*ucgaAuuuaUuAcAggu*g*c 349 AD00350 AD00350-SS g*c*agauacAuCuCcaaguau*a(GLX-n) 301 AD00350-AS u*A*uacuUggagAuGuAucu*g*c 350 AD00351 AD00351-SS g*c*caaugaCuUuGacgagua*a(GLX-n) 302 AD00351-AS u*U*acucGucaaAgUcAuug*g*c 351 AD00385 AD00385-SS (GLS-5)*(Invab)*guaucggaUcUuCaccgucaa*(Invab) 303 AD00385-AS u*U*gaCg(gUNA)ugaaGaUcCgau*a*c 352 AD00386 AD00386-SS (GLS-5)*(Invab)*cgaaugccCuUuGaccucaua*(Invab) 304 AD00386-AS u*A*ugagGucaaAgGgCauu*c*g 353 AD00387 AD00387-SS (GLS-5)*(Invab)*ggucaacuUcCuCcugcgaaa*(Invab) 305 AD00387-AS u*U*ucGc(aUNA)ggagGaAgUuga*c*c 354 AD00388 AD00388-SS (GLS-5)*(Invab)*ggcuacuaCaCcUaccugaua*(Invab) 306 AD00388-AS u*A*ucagGuaggUgUaGuag*c*c 355 AD00389 AD00389-SS (GLS-5)*(Invab)*cuugcagaAgAgAacaucgua*(Invab) 307 AD00389-AS u*A*cgauGuucuCuUcUgca*a*g 356 AD00390 AD00390-SS (GLS-5)*(Invab)*guacuccaCaAuCcagccuua*(Invab) 308 AD00390-AS u*A*agGc(uUNA)ggauUgUgGagu*a*c 357 AD00391 AD00391-SS (GLS-5)*(Invab)*caccacagCcAaAgauaagaa*(Invab) 309 AD00391-AS u*U*cuuaUcuuuGgCuGugg*u*g 358 AD00392 AD00392-SS (GLS-5)*(Invab)*gguuccaaGcCuUggcucaaa*(Invab) 310 AD00392-AS u*U*ugAg(cUNA)caagGcUuGgaa*c*c 359 AD00393 AD00393-SS (GLS-5)*(Invab)*cuuccaagCcUuGgcucaaua*(Invab) 311 AD00393-AS u*A*uugaGccaaGgCuUgga*a*g 360 AD00394 AD00394-SS (GLS-5)*(Invab)*cgagaccaGgAuGccacuaua*(Invab) 312 AD00394-AS u*A*uaguGgcauCcUgGucu*c*g 361 AD00395 AD00395-SS (GLS-5)*(Invab)*gaaguaugAgCuGgacaaaga*(Invab) 313 AD00395-AS u*C*uuugUccagCuCaUacu*u*c 362 AD00396 AD00396-SS (GLS-5)*(Invab)*cucuagcuUuCaAaguucaca*(Invab) 314 AD00396-AS u*G*ugaaCuuugAaAgCuag*a*g 363 AD00397 AD00397-SS (GLS-5)*(Invab)*gagcugucCaAuGacuuugaa*(Invab) 315 AD00397-AS u*U*caaaGucauUgGaCagc*u*c 364 AD00410 AD00410-SS (GLS-5)*(Invab)*cgccuuugUcAuCuucgggaa*(Invab) 316 AD00410-AS u*U*cccgAagauGaCaAagg*c*g 365 AD00411 AD00411-SS (GLS-5)*(Invab)*cauccccaUcGuGaccucuca*(Invab) 317 AD00411-AS u*G*agagGucacGaUgGgga*u*g 366 AD00412 AD00412-SS (GLS-5)*(Invab)*cucacaguAaUgCaggacuua*(Invab) 318 AD00412-AS u*A*agucCugcaUuAcUgug*a*g 367 AD00413 AD00413-SS (GLS-5)*(Invab)*cucucucuGgCuGucaaccua*(Invab) 319 AD00413-AS u*A*gguuGacagCcAgAgag*a*g 368 AD00414 AD00414-SS (GLS-5)*(Invab)*guuaacaaAuUuCugaccaca*(Invab) 320 AD00414-AS u*G*ugguCagaaAuUuGuua*a*c 369 AD00415 AD00415-SS (GLS-5)*(Invab)*gcuacugcAgCuAaaagacua*(Invab) 321 AD00415-AS u*A*gucuUuuagCuGcAgua*g*c 370 AD00416 AD00416-SS (GLS-5)*(Invab)*gucaaugaAcAgAgauacuaa*(Invab) 322 AD00416-AS u*U*aguaUcucuGuUcAuug*a*c 371 AD00417 AD00417-SS (GLS-5)*(Invab)*gaugauccUuGaGaucuguaa*(Invab) 323 AD00417-AS u*U*acagAucucAaGgAuca*u*c 372 AD00418 AD00418-SS (GLS-5)*(Invab)*guuggacaUaUcCaugaugaa*(Invab) 324 AD00418-AS u*U*caucAuggaUaUgUcca*a*c 373 AD00419 AD00419-SS (GLS-5)*(Invab)*guguccaaUgAcUuugacgaa*(Invab) 325 AD00419-AS u*U*cgucAaaguCaUuGgac*a*c 374 AD00420 AD00420-SS (GLS-5)*(Invab)*ccuuugacGaGuAcaucauga*(Invab) 326 AD00420-AS u*C*augaUguacUcGuCaaa*g*g 375 AD00526 AD00526-SS (GLS-15)*(Invab)*gugugcauUaCcUggaugaaa*(Invab) 327 AD00526-AS u*U*ucauCcaggUaAuGcac*a*c 376 AD00527 AD00527-SS (GLS-15)*(Invab)*gugcagcuAaAaGacuuugaa*(Invab) 328 AD00527-AS u*U*caaaGucuuUuAgCugc*a*c 377 AD00528 AD00528-SS (GLS-15)*(Invab)*ccucaaugAaCaGagauacua*(Invab) 329 AD00528-AS u*A*guauCucugUuCaUuga*g*g 378 AD00529 AD00529-SS (GLS-15)*(Invab)*gaaugaacAgAgAuacuacga*(Invab) 330 AD00529-AS u*C*guagUaucuCuGuUcau*u*c 379 AD00530 AD00530-SS (GLS-15)*(Invab)*ggacaaugUaCcAugcuaaga*(Invab) 331 AD00530-AS u*C*uuagCauggUaCaUugu*c*c 380 AD00531 AD00531-SS (GLS-15)*(Invab)*ggccaagaAcAcUaugaucca*(Invab) 332 AD00531-AS u*G*gaucAuaguGuUcUugg*c*c 381 AD00532 AD00532-SS (GLS-15)*(Invab)*cuggacauAuCcAugaugaca*(Invab) 333 AD00532-AS u*G*ucauCauggAuAuGucc*a*g 382 AD00533 AD00533-SS (GLS-15)*(Invab)*gggucucaCaCuCugaggaua*(Invab) 334 AD00533-AS u*A*uccuCagagUgUgAgac*c*c 383 AD00534 AD00534-SS (GLS-15)*(Invab)*ccuuuaauGuAgAgcuuauca*(Invab) 335 AD00534-AS u*G*auaaGcucuAcAuUaaa*g*g 384 AD00535 AD00535-SS (GLS-15)*(Invab)*gaggagugGaCuAuguguaca*(Invab) 336 AD00535-AS u*G*uacaCauagUcCaCucc*u*c 385 AD00579 AD00579-SS (GLS-15)*(Invab)*cauccgagCcUaCuaugaaaa*(Invab) 337 AD00579-AS u*U*uucaUaguaGgCuCgga*u*g 386 AD00580 AD00580-SS (GLS-15)*(Invab)*caauucuaCuAcAucuauaaa*(Invab) 338 AD00580-AS u*U*uauaGauguAgUaGaau*u*g 387 AD00581 AD00581-SS (GLS-15)*(Invab)*cuucggcUgAaUaagaagaa*(Invab) 339 AD00581-AS u*U*cuucUuauuCaGcAcga*a*g 388 AD00582 AD00582-SS (GLS-15)*(Invab)*gccuacucUgUuGuucgaaaa*(Invab) 340 AD00582-AS u*U*uucgAacaaCaGaGuag*g*c 389 AD00583 AD00583-SS (GLS-15)*(Invab)*ggcagcuaAaAgAcuuugaca*(Invab) 341 AD00583-AS u*G*ucaaAgucuUuUaGcug*c*c 390 AD00584 AD00584-SS (GLS-15)*(Invab)*gcucaaggUcAcCauaaaaca*(Invab) 342 AD00584-AS u*G*uuuuAugguGaCcUuga*g*c 391 AD00740 AD00740-SS (GLS-15)*(Invab)*cuaaugcaGgAcuUcuucau*a*(Invab) 392 AD00740-AS u*A*ugAagaaguCcUgcaUu*a*g 407 AD00741 AD00741-SS (GLS-15)*(Invab)*gaugcaggAcUucUucaucg*a*(Invab) 393 AD00741-AS u*C*gaUgaagaaGuCcugCa*u*c 408 AD00742 AD00742-SS (GLS-15)*(Invab)*guacccuaCuCugUuguucg*a*(Invab) 394 AD00742-AS u*C*gaAcaacagAgUaggGu*a*c 409 AD00743 AD00743-SS (GLS-15)*(Invab)*cacucuguUgUucGaaacga*a*(Invab) 395 AD00743-AS u*U*cgUuucgaaCaAcagAg*u*g 410 AD00744 AD00744-SS (GLS-15)*(Invab)*ggaaacgaGcAggUggaaau*a*(Invab) 396 AD00744-AS u*A*uuUccaccuGcUcguUu*c*c 411 AD00344-1 AD00344-1-SS (GLS-15)*(Invab)*cgagaacaUcGuUucccgaa*a*(Invab) 397 AD00344-1-AS u*U*ucGg(gUNA)aaacGaUgUucu*c*g 412 AD00388-1 AD00388-1-SS (GLS-15)*(Invab)*ggcuacuaCaCcUaccugau*a*(Invab) 398 AD00388-1-AS u*A*ucagGuaggUgUaGuag*c*c 413 AD00389-1 AD00389-1-SS (GLS-15)*(Invab)*cuugcagaAgAgAacaucgu*a*(Invab) 399 AD00389-1-AS u*A*cgauGuucuCuUcUgca*a*g 414 AD00385-1 AD00385-1-SS (GLS-15)*(Invab)*guaucggaUcUuCaccgucaa*(Invab) 400 AD00385-1-AS u*U*gaCg(gUNA)ugaaGaUcCgau*a*c 415 AD00416-1 AD00416-1-SS (GLS-15)*(Invab)*gucaaugaAcAgAgauacuaa*(Invab) 401 AD00416-1-AS u*U*aguaUcucuGuUcAuug*a*c 416 AD00580-1 AD00580-1-SS (GLS-15)*(Invab)*caauccuaCuAcAucuauaa*a*(Invab) 402 AD00580-1-AS u*U*uauaGauguAgUaGaau*u*g 417 AD00343-1 AD00343-1-SS (GLS-15)*(Invab)*gcucuaacAaUuAccugcau*a*(Invab) 403 AD00343-1-AS u*A*ugCa(gUNA)guaaUuGuUgga*g*c 418 AD00388-1 AD00388-1-SS (GLS-15)*(Invab)*ggcuacuaCaCcUaccugau*a*(Invab) 404 AD00388-1-AS u*A*ucagGuaggUgUaGuag*c*c 419 AD00581-1 AD00581-1-SS (GLS-15)*(Invab)*cuucggcUgAaUaagaaga*a*(Invab) 405 AD00581-1-AS u*U*cuucUuauuCaGcAcga*a*g 420 AD00389-1 AD00389-1-SS (GLS-15)*(Invab)*cuugcagaAgAgAacaucgu*a*(Invab) 406 AD00389-1-AS u*A*cgauGuucuCuUcUgca*a*g 421 Mismatch

It is known to those skilled in the art that mismatches are tolerated for the efficacy of dsRNA, especially if the mismatch is within the terminal region of the dsRNA. Certain mismatches are better tolerated, such as those with wobble base pairs G:U and A:C (Du et el., 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 invention, the C3 dsRNA agent may contain one or more mismatches to the C3 target sequence. In some embodiments, the C3 dsRNA reagents of the invention contain no mismatches. In certain embodiments, C3 dsRNA reagents of the invention contain no more than 1 mismatch. In some embodiments, the C3 dsRNA reagents of the invention contain no more than 2 mismatches. In certain embodiments, C3 dsRNA reagents of the invention contain no more than 3 mismatches. In some embodiments of the invention, the antisense strand of the C3 dsRNA agent contains a mismatch to the C3 target sequence that is not centered in the complementary region. In some embodiments, the antisense strand of the C3 dsRNA agent contains 1, 2, 3, 4, or more mismatches located at the last 5, 4 of one or both of the 5' or 3' ends of the complementary region , 3, 2 or 1 nucleotide. The methods described herein and/or methods known in the art can be used to determine whether a C3 dsRNA agent comprising a mismatch to a C3 target sequence is effective in inhibiting expression of the C3 gene. complementarity

As used herein, unless otherwise stated, the term "complementarity/complementarity" is used when describing a first nucleotide sequence (e.g., C3 dsRNA agent sense strand or target C3 mRNA) to a second nucleotide sequence (e.g., C3 dsRNA reagent antisense strand or single-stranded antisense polynucleotide) refers to an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide containing a second nucleotide sequence. The ability of an acid or polynucleotide to hybridize [form hydrogen bonds between base pairs under mammalian physiological conditions (or similar conditions in vitro)] and, under certain conditions, to form a double helix or duplex structure. Other conditions may also apply, such as physiologically relevant conditions that may be encountered in living organisms. The skilled person will be able to determine the set of conditions most suitable 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, so long as they are at least to the extent required for hybridization as described above. . Sequence identity or complementarity is independent of modification.

For example, a complementary sequence within a C3 dsRNA as described herein includes an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence in one or base pairing over the entire length of two nucleotide sequences. Such sequences may be referred to herein as "completely complementary" to each other. It will 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 herein to be mismatches based on complementarity. For example, the C3 dsRNA reagent contains one oligonucleotide that is 19 nucleotides in length and another oligonucleotide that is 20 nucleotides in length, with the longer oligonucleotide containing the same oligonucleotide as the shorter oligonucleotide. A sequence of 19 nucleotides whose nucleotides are completely complementary, which for the purposes described herein may be referred to as "perfectly complementary." Thus, as used herein, "perfect complementarity" means that all (100%) 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. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

As used herein, the term "substantially complementary" means that in hybrid pairs of nucleobase sequences, at least about 85% (but not all) of the bases in the contiguous sequence of the first polynucleotide will be with the second polynucleotide. The same number of bases in a contiguous sequence of nucleotides hybridize. If two sequences contain one or more mismatched base pairs when hybridized, for example at least 1, 2, 3, 4 or 5 mismatched base pairs, the term "substantially complementary" may be used to refer to the first The sequence 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 body while retaining the ability to hybridize under conditions most relevant to its end application, e.g., inhibition of C3 gene expression via the RISC pathway. The term "partially complementary" may be used herein to refer to a hybrid pair of nucleobase sequences in which at least 75% (but not all) of the bases in the contiguous sequence of a first polynucleotide will be with those of a second polynucleotide. Hybridization of the same number of bases in a contiguous sequence. In some embodiments, "partially complementary" refers to 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 will match the Two polynucleotides hybridize to the same number of bases in a contiguous sequence.

The terms "complementary," "completely complementary," "substantially complementary," and "partially complementary" when used herein may be used to refer to the base matching between the sense strand and the antisense strand of the C3 dsRNA reagent, the antisense strand of the C3 dsRNA reagent A base match between the strand and the sequence of the target C3 mRNA, or a base match between a single-stranded antisense oligonucleotide and the sequence of the target C3 mRNA. It will be understood that the term "antisense strand of a C3 dsRNA agent" may refer to the same sequence as a "C3 antisense polynucleotide agent."

As used herein, the terms "substantially identical" or "substantially identical" when referring to a nucleic acid sequence means that the nucleic acid sequence contains a sequence that has at least about 85% or greater sequence identity compared to a reference sequence, 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% identical. Percent sequence identity is determined by comparing the best alignment of two sequences over an alignment window. The percentage is calculated by determining the number of positions where the same nucleic acid base occurs in both sequences to yield the number of matching positions; dividing the number of matching positions by the total number of positions in the alignment window, and multiplying the result by 100, resulting in percent sequence identity. The inventions disclosed herein include nucleotide sequences that are substantially identical to those disclosed herein (eg, in Tables 1-3). In some embodiments, the nucleotide sequence is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90% identical to, a sequence disclosed herein (e.g., in Tables 1-3). %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referred to using standard nucleotide nomenclature. As used herein, the term "double-stranded RNA" or "dsRNA" refers to a sequence comprising an RNA molecule or a complex of RNAi molecules having a hybridized double-stranded nucleic acid strand comprising two antiparallel and substantially or completely complementary nucleic acid strands. Strand regions, which are referred to as having "sense" and "antisense" orientations relative to the target C3 RNA, respectively. The double-stranded region can be of any desired length that allows 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. Considering duplexes between 9 and 30 base pairs, duplexes can be any length within this range, e.g., 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 subrange 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, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 bases pair, 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 reagents produced in cells by processing with Dicer and similar enzymes is typically in the range of 19-22 base pairs. One strand of the double-stranded region of the C3 dsDNA agent contains a sequence that is substantially complementary to a region of the target C3 RNA. The two strands forming a duplex structure can come from a single RNA molecule with at least one self-complementary region, or can be formed from two or more separate RNA molecules. In the case where the double-stranded region is formed from a single molecule, the molecule may have a duplex structure formed by one strand of the single-stranded nucleotide strand at the 3'-end and the corresponding other strand at the 5'-end (herein called a "hairpin loop"). In some embodiments of the invention, the hairpin configuration includes 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 the C3 dsRNA reagent consist of separate RNA molecules, these molecules need not be covalently linked, but may be covalently linked. When two strands are covalently connected by means other than a hairpin loop, the connecting structure is called a "linker." The term "siRNA" is also used herein to refer to a dsRNA agent as described herein.

In some embodiments of the invention, a C3 dsRNA reagent can comprise sense and antisense sequences with unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA reagent. Ends without unpaired nucleotides are called "blunt ends" and have no nucleotide overhangs. If both ends of the dsRNA reagent are blunt-ended, the dsRNA is said to be "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, the C3 dsRNA Both ends of the reagent are blunt-ended.

In some embodiments of the dsRNA reagents of the 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 strand of the dsRNA reagent. For example, nucleotide overhangs exist when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, and vice versa. dsRNA can contain overhangs of at least 1, 2, 3, 4, 5, 6, or more nucleotides. Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides. It will be understood that in some embodiments, the nucleotide overhangs are 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 overhangs may be present on the antisense strand of the dsRNA reagent. sense strand or the 5' end, 3' end or both ends of the sense strand. In certain embodiments of the invention, one or more nucleotides in the overhang are replaced with nucleoside phosphorothioates.

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

In some embodiments of the invention, the RNA of the C3 RNAi agent is chemically modified to obtain enhanced stability and/or one or more other beneficial properties. Nucleic acids in certain embodiments of the invention can be synthesized and/or modified by methods well known in the art, for example, see "Current protocols in Nucleic Acid Chemistry," Beaucage, S. L. et al. (Eds.), John Wiley & Sons , Inc., New York, N.Y., USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of the C3 dsRNA reagents of the invention include, for example: (a) terminal modifications, such as 5' end modifications (phosphorylation, conjugation, reverse ligation, etc.), 3' end modifications (conjugation; fusion, DNA nucleotides, reverse ligation, etc.); (b) base modifications, such as base replacement, missing bases with stabilizing bases, destabilizing bases or base pairing with an expanded partner library ( abasic nucleotides) or conjugated bases; (c) sugar modifications (e.g., at the 2' position or 4' position) or substitution of sugars; and (d) backbone modifications, including modification of phosphodiester bonds or Replace. Specific examples of RNA compounds useful in certain embodiments of the C3 dsRNA reagents, C3 antisense polynucleotides, and C3 sense polynucleotides of the invention include, but are not limited to, RNAs containing modified backbones or without natural internucleoside linkages. . As a non-limiting example, RNA with backbone modifications may not have phosphorus atoms in the backbone. RNA that does not have a phosphorus atom in its internucleoside backbone is called an oligonucleotide. In certain embodiments of the invention, the modified RNA has a phosphorus atom in its internucleoside backbone.

It will 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 species as described herein. Ribonucleotide/ribonucleoside analogs or derivatives described above or known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules may be modified in the nucleobase structure or ribose-phosphate backbone structure (eg, 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 comprise at least one modified ribonucleoside, including, but not limited to, 2'-O-methyl modified nucleosides, nucleosides containing a 5' phosphorothioate group, and Terminal nucleosides linked to cholesterol derivatives or dodecanoic acid didecamide groups, locked nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino modified nucleosides Nucleosides, 2'-alkyl modified nucleosides, morpholino nucleosides, phosphoramidates, or unnatural bases containing nucleosides, or any combination thereof. In some embodiments of the invention, the RNA molecule contains 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 full length of the ribonucleoside of the C3 dsRNA reagent molecule. The modifications need not be the same for each of the multiple modified ribonucleosides in such an RNA molecule.

In some embodiments, the dsRNA reagents, C3 antisense polynucleotides and/or C3 sense polynucleotides of the invention may comprise one or more independently selected modified nucleotides and/or one or more independently selected of non-phosphodiester bonds. As used herein, the term "independently selected" is used to refer to selected elements, such as modified nucleotides, non-phosphodiester bonds, etc., meaning that two or more selected elements may be identical to each other but need not be identical to each other. As used herein, a "nucleotide base", "nucleotide" or "nucleobase" is a heterocyclic pyrimidine or purine compound that is a standard component of all nucleic acids and includes the bases that form nucleotides: adenine Purine (a), guanine (g), cytosine (c), thymine (t) and uracil (u). Nucleobases may be further modified to include, but are not intended to be limited to: universal bases, hydrophobic bases, hybrid bases, size-enlarged bases, and fluorinated bases. The term "ribonucleotide" or "nucleotide" may be used herein to refer to unmodified nucleotides, modified nucleotides, or alternative moieties. One skilled in the art will recognize that guanine, cytosine, adenine and uracil can be replaced by other moieties without significantly altering the base pairing of oligonucleotides containing nucleotides with such replaced moieties characteristic.

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

Modified RNA backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkyl phosphonates (including 3'- Alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkyl phosphoramidates), thiophosphates, thioalkyl Phosphonates, thioalkyl phosphate triesters, and boronic acid phosphates (which have the normal 3'-5' linkage, as well as the 2'-5' linkage analogs of these, and those with inverted polarity, where adjacent Pairs of nucleoside units are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' format). Also included are various salts, mixed salts and free acid forms. Methods for preparing phosphorus-containing bonds are routine practices in the art, and such methods can be used to prepare certain modified C3 dsRNA reagents, certain modified C3 antisense polynucleotides, and/or certain modified C3 of the invention Sense polynucleotide.

Modified RNA backbones that do not contain a phosphorus atom have structures consisting of short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chains The backbone formed by the linkages between heteroatoms or heterocyclic nucleosides. They include those with morpholine bonds (formed in part from the sugar moiety of the nucleoside); siloxane skeletons; sulfide, triacetyl and triacetyl skeletons; methylacetyl and thiomethylacetyl skeletons; methylenemethacetyl and thiomethylacetyl skeletons Skeletons; skeletons containing olefins; sulfamate skeletons; methyleneimino and methylenehydrazino skeletons; sulfonate and sulfonamide skeletons; amide skeletons; and others mixed with N, O, S and CH ₂ ingredient portion. Methods of preparing modified RNA backbones that do not contain phosphorus atoms are routine practice in the art, and such methods can be used to prepare certain modified C3 dsRNA reagents, certain modified C3 antisense polynucleotides, and/or the present invention. Or certain modified C3 sense polynucleotides.

In certain embodiments of the invention, RNA mimetics are included in C3 dsRNA, C3 antisense polynucleotides, and/or C3 sense polynucleotides, such as, but not limited to, sugars that replace nucleotide units with new groups. and internucleoside linkages (i.e. backbone). In such embodiments, base units are maintained for hybridization to the appropriate C3 nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobase is retained and bound directly or indirectly to the aza nitrogen atom of the amide moiety of the backbone. Methods of preparing RNA mimetics are routinely practiced in the art, and such methods can be used to prepare certain modified C3 dsRNA agents of the invention.

Some embodiments of the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly _-CH2- NH- _CH2- , _-CH2 -N( _CH3 )-O-CH ₂ -[called methylene (methylimino) or MMI skeleton], -CH ₂ -ON(CH ₃ )-CH ₂ -, -CH ₂ -N(CH ₃ )-N(CH ₃ )-CH ₂ - and -N(CH ₃ )-CH ₂ - [where the natural phosphodiester backbone is represented as -OPO-CH ₂ -]. Methods for preparing RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare certain modified C3 dsRNA reagents, certain C3 antisenses of the invention polynucleotides and/or certain C3 sense polynucleotides.

Modified RNA may also contain one or more substituted sugar moieties. The C3 dsRNA, C3 antisense polynucleotide and/or C3 sense polynucleotide of the present invention may contain 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, where alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl. Exemplary suitable modifications include: O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH 2 ) _n NH ₂ , O( _{CH 2 ) n} CH ₃ , O( CH ₂ ) _n ONH ₂ , and O(CH ₂ ) _n ON [(CH ₂ ) _n CH ₃ )] ₂ , where n and m range from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl , SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl , aminoalkylamino, polyalkylamino; substituted silyl group, RNA cleavage group, reporter group, intercalator; groups used to improve the pharmacokinetic properties of C3 dsRNA reagents; or used to improve C3 groups with pharmacodynamic properties of dsRNA reagents, C3 antisense polynucleotides and/or C3 sense polynucleotides, and other substituents with similar properties. In some embodiments, modifications include 2'-O-CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2' -MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), that is, alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminoethoxyethoxy, the 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 of preparing modified RNAs such as those described are routinely practiced in the art, and such methods may be used to prepare certain modified C3 dsRNA agents of the invention.

Other modifications include 2'-methoxy (2'-OCH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the C3 dsRNA reagents of the invention, the RNA of the C3 antisense polynucleotide, the C3 sense polynucleotide and/or other positions of the C3 sense polynucleotide, particularly the 3' terminal nucleoside The 3' position of the sugar on the acid or 2'-5' linked C3 dsRNA, C3 antisense polynucleotide, or C3 sense polynucleotide, and the 5' position of the 5' terminal nucleotide. The C3 dsRNA reagent, C3 antisense polynucleotide, and/or C3 sense polynucleotide may also have a sugar mimetic, such as a cyclobutyl moiety in place of the pentofuranose. Methods of 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 invention .

In some embodiments, C3 dsRNA agents, C3 antisense polynucleotides, and/or C3 sense polynucleotides can include nucleobase (often referred to as "bases" in the art) 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-thiocytosan Pyrimidine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil ; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine; 5-halo, especially 5-bromo, 5- Trifluoromethyl and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-azaguanine and 7-azaadenine and 3-azaguanine and 3-azaadenine. Additional nucleobases that may be included in certain embodiments of the C3 dsRNA reagents 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, J. L, 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, S. T. and Lebleu, B., Ed., CRC Press, 1993. Methods of preparing dsRNA, C3 antisense strand polynucleotides, and/or C3 sense strand polynucleotides (such as those described herein) comprising nucleobase modifications and/or substitutions are routinely practiced in the art, and such methods Certain modified C3 dsRNA reagents, C3 sense polynucleotides and/or C3 antisense polynucleotides can be used to prepare the present invention.

Certain embodiments of the C3 dsRNA reagents, C3 antisense polynucleotides, and/or C3 sense polynucleotides of the invention include RNA modified to include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" ribose in the 3'-endostructural conformation. Adding locked nucleic acid to the C3 dsRNA reagent, C3 antisense polynucleotide and/or C3 sense polynucleotide of the present invention can increase the 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 of preparing dsRNA reagents, C3 antisense polynucleotides, and/or C3 sense polynucleotides containing locked nucleic acids are routinely practiced in the art, and such methods may be used to prepare certain modified C3 dsRNA reagents of the invention. Certain embodiments of the C3 dsRNA compounds, sense polynucleotides and/or antisense polynucleotides of the invention include at least one modified nucleotide, 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, 2'-F-arabinose nucleotides , 2'-methoxyethyl nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide and 3'-Ome nucleotide, including Nucleotides with a 5'-phosphorothioate group, or terminal nucleotides linked to cholesterol derivatives or didecylamide dodecanoate groups, 2'-amino modified nucleotides, phosphoramidates or contain unnatural bases of nucleotides. In some embodiments, the C3 dsRNA compound contains an E-vinylphosphonate nucleotide at the 5' end of the antisense strand (also referred to herein as the guide strand).

In certain embodiments of the 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 at least one modification The nucleotides include: abasic nucleotides, ribitol, reverse nucleotides, reverse abasic nucleotides, reverse 2'-OMe nucleotides, reverse 2'-deoxy nucleotides . It is known to those skilled in the art that the inclusion of abasic or reverse abasic nucleotides at the termini of oligonucleotides 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).

In certain embodiments of the invention, C3 dsRNA compounds, antisense polynucleotides comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises open nucleic acid nucleotides (UNA) or/and Glycol nucleic acid nucleotides (GNA). Those skilled in the art know that UNA and GNA are thermally unstable chemical modifications that can significantly improve the off-target profile 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).

In certain embodiments, the invention relates to open nucleic acid (UNA) oligomers for use in therapy. Unlocked 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 shown that incorporation of UNA is well tolerated by siRNA gene silencing activity and in some cases can even enhance its activity (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 ).

UNA is a heat-labile modification, and replacing ribonucleotides with UNA will reduce 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). miRNA mainly recognizes the target gene through base pairing between the antisense seed region (positions 2-8 starting from the 5' end) and the target mRNA for gene suppression. 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 a miRNA-mediated mechanism. Therefore, adding 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 complexes of RNA oligonucleotides contain at least one UNA nucleotide 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 ).

According to this technical solution, potential advantages of incorporating UNA in RNA oligonucleotides or complexes of RNA oligonucleotides include, but are not limited to:

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

2. UNA is well tolerated in terms of siRNA activity. In some cases, UNA can lead to increased activity.

Exemplary UNA monomers that can be used in this technical solution include, but are not limited to: .

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

Another modification may be included in the RNA of the C3 dsRNA agent, C3 antisense polynucleotide, and/or C3 sense polynucleotide of certain embodiments of the invention, including enhancing the C3 dsRNA agent, C3 antisense polynucleotide, respectively. and/or one or more ligands, moieties, or conjugates chemically linked to RNA of one or more features of the C3 sense polynucleotide. 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 the C3 dsRNA agent, pharmacokinetic properties of the C3 dsRNA agent, and Cellular uptake of C3 dsRNA reagent. In some embodiments of the invention, the C3 dsRNA reagents comprise one or more targeting or linking groups, which, in certain embodiments of the invention, are conjugated to the sense strand. Non-limiting examples of targeting groups are compounds containing N-acetyl-galactosamine (GalNAc). The terms "targeting group", "targeting agent", "linker", "targeting compound" and "targeting ligand" are used interchangeably herein. In certain embodiments of the invention, the C3 dsRNA agent comprises a targeting compound conjugated to the 5'-end of the sense strand. In certain embodiments of the invention, the C3 dsRNA agent comprises a targeting compound conjugated to the 3'-end of the sense strand. In some embodiments of the invention, the C3 dsRNA agent comprises a GalNAc-containing targeting group. In certain embodiments of the invention, the C3 dsRNA agent does not comprise a targeting compound conjugated to one or both of the 3'-end and 5'-end of the sense strand. In certain embodiments of the invention, the C3 dsRNA agent does not comprise a GalNAc-containing targeting compound conjugated to one or both of the 5'-end and the 3'-end of the sense strand.

Additional targeting and linking agents are well known in the art and, for example, targeting and linking agents useful in certain embodiments of the present invention include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. . Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl- S-Tritylmercaptan (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biog. Med. Chem. Let., 1993, 3:2765- 2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains such as dodecanediol 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 triethylammonium 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), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or Adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmitate moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or octadecylamine or Hexamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

Certain embodiments of compositions comprising C3 dsRNA agents, C3 antisense polynucleotides, and/or C3 sense polynucleotides may include ligands that alter the distribution, targeting, etc. properties of the C3 dsRNA agent. In some embodiments of compositions comprising a C3 dsRNA agent of the invention, e.g., the ligand increases the response to a selected target (e.g., molecule, cell or cell type, compartment, e.g., cell) compared to a species in which such ligand is not present. or organ compartment, tissue, organ or body region) affinity. Ligands useful in the compositions and/or methods of the invention may be naturally occurring substances such as proteins (e.g. human serum albumin (HSA), low density lipoprotein (LDL) or globulin), carbohydrates (e.g. , dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids or polyamines. Examples of polyamino acids are polylysine (PLL), polyL-aspartic acid, polyL-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 polyphosphazine. Examples of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendritic polyamines, arginine, amidine , protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines or α-helical peptides.

Ligands comprised in the compositions and/or methods of the present invention may comprise targeting groups, non-limiting examples of which are cell or tissue targeting agents such as lectins, glycoproteins, lipids or proteins, e.g. binding to specific cells Types of antibodies such as kidney cells or liver cells. Targeting groups can be thyrotropin, melanogen, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N- Acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent 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.

Other examples of ligands include dyes, intercalators (e.g., acridines), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazines) , dihydrophenazine); artificial endonucleases (such as EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (ten Hexadecyl)glycerin, geranyloxyhexyl, cetylglycerin, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O3-(oleyl)lithogallstone Acids, O3-(oleyl)cholic acid, dimethoxytrityl or phenoxazine and peptide conjugates (e.g., antennal peptide, Tat peptide), alkylating agents, phosphates, amino groups, thiol groups, PEG (e.g., PEG-40K), MPEG, [MPEG] ₂ , polyamino, alkyl, substituted alkyl, radiolabel, enzyme, hapten (e.g., biotin), transport/absorption enhancer (e.g., aspirin, vitamin E, folic acid), synthetic ribonuclease (such as imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, Eu ³⁺ complex of tetraaza macrocycle), dinitrophenyl, HRP or AP.

Ligands included in the compositions and/or methods of the present invention may be proteins, such as glycoproteins or peptides, such as molecules with specific affinity for the co-ligand, or antibodies, such as with specific cell types such as cancer cells, endothelial cells, Antibodies that bind to cardiomyocytes or bone cells. Ligands useful in embodiments of the compositions and/or methods of the invention may be hormones or hormone receptors. Ligands useful in embodiments of the compositions and/or methods of the invention may be lipids, lectins, carbohydrates, vitamins, coenzymes, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose or polyvalent fucose. Ligands useful in embodiments of the compositions and/or methods of the invention may be agents that increase C3 dsRNA, for example, by disrupting the cell's cytoskeleton (e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments) Substances taken up into cells. Non-limiting examples of such agents are: paclitaxel, vincristine, vinblastine, cytochalasin, nocodazole, microfilament polymerization agent, erythrin A, phalloidin, pontolide A, Indanocristine and troponin.

In some embodiments, ligands linked to the C3 dsRNA agents of the invention serve as pharmacokinetic (PK) modulators. Examples of PK modulators useful in the compositions and methods of the present invention include, but are not limited to: lipophilic agents, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, bile acids, Lithocholic acid, dialkyl glycerides, digyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, aptamers that bind to serum proteins, etc. Oligonucleotides containing many phosphorothioate linkages are also known to bind to serum proteins, therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g. about 5 bases, 10 bases Base, 15 base, or 20 base oligonucleotides may also be used as ligands in the compositions and/or methods of the invention. C3 dsRNA Reagent Composition

In some embodiments of the invention, a C3 dsRNA agent is in the composition. Compositions of the invention may comprise one or more C3 dsRNA agents and optionally one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc., according to some implementations of the methods of the invention Non-limiting examples of targeting agents that may be used in the protocol are agents that direct the C3 dsRNA agents of the invention to and/or into the 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 invention, it may be desirable to target C3 dsRNA agents to and/or enter hepatocytes. It will be appreciated that in some embodiments of the methods of the invention, the therapeutic agent comprises a C3 dsRNA agent with 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 GalNAc-containing delivery compound and included in a composition containing a pharmaceutically acceptable carrier, and in the absence of any detectable label linked to the C3 dsRNA agent or Targeting agents, etc. are administered to cells or subjects.

Where the C3 dsRNA reagents of the present invention are administered together with and/or linked 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 reagents used in the method of the present invention. Labeling reagents can be used in certain methods of the invention to determine the location of C3 dsRNA agents in cells and tissues, and can be used to identify cells, tissues that have been administered therapeutic compositions containing C3 dsRNA agents in the methods of the invention. or organ location. Means for attaching and using labeling reagents such as enzyme labels, dyes, radioactive labels, etc. are well known in the art. It will be appreciated that in some embodiments of the compositions and methods of the invention, the labeling agent is linked to one or both of the sense and antisense polynucleotides comprised in the C3 dsRNA agent. Delivery of C3 dsRNA Reagents and C3 Antisense Polynucleotide Reagents

Certain embodiments of the methods of the invention include delivering a C3 dsRNA agent into the cell. As used herein, the term "delivery" means promoting or affecting cellular uptake or absorption. Absorption or uptake of C3 dsRNA agents can occur by separate diffusion or active cellular processes, or by the use of delivery agents, targeting agents, etc., which can be associated with the C3 dsRNA agents of the invention. Modes of delivery suitable for use in 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 invention, the C3 dsRNA agent is linked to the delivery agent.

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 used successfully in the art to deliver therapeutic RNAi agents to treat a variety of diseases and conditions, such as, but not limited to: liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, etc. . Detailed information on various delivery methods can be found in publications such as: Nikam, R.R. & K. R. Gore (2018) Nucleic Acid Ther, 28 (4), 209-224 Aug 2018; Springer A.D. & 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, J.K. et al., (2014) J. Am. Chem. Soc. 136:16958-16961, the contents of which are incorporated herein by reference.

Some embodiments of the invention include the use of lipid nanoparticles (LNPs) to deliver the C3 dsRNA agents of the invention to cells, tissues and/or subjects. LNPs are commonly used to deliver C3 dsRNA agents in vivo, including therapeutic C3 dsRNA agents. One benefit of using LNP or other delivery agents is the increased stability of the C3 RNA agent when delivered to a subject using LNP or other delivery agents. In some embodiments of the invention, the LNPs comprise cationic LNPs loaded with one or more C3 RNAi molecules of the invention. LNPs containing C3 RNAi molecules are administered to the subject. The LNPs and their attached C3 RNAi molecules are taken up by cells via endocytosis, and their presence results in the release of RNAi triggering molecules, thereby mediating RNAi.

Another non-limiting example of a delivery agent that may be used in embodiments of the invention to deliver the C3 dsRNA agent of the invention to cells, tissues and/or subjects is an agent comprising GalNAc linked to the C3 dsRNA agent of the invention and delivering C3 dsRNA reagents to cells, tissues, and/or subjects. Examples of certain other GalNAc-containing delivery agents that may be used in certain embodiments of the methods and compositions of the invention are disclosed in PCT application WO2020191183 A1. Non-limiting examples of GalNAc targeting ligands that can be used in the compositions and methods of the invention to deliver C3 dsRNA agents to cells are targeting ligand clusters. Examples of targeting ligand clusters proposed here are: GalNAc ligands with phosphodiester linkages (GLO) and GalNAc ligands with phosphorothioate linkages (GLS). The term "GLX-n" may be used herein to refer to attached GalNAC-containing compounds. Examples are, but are 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 is shown below. In the figure below, the connection position of the GalNAc targeting ligand and the RNAi agent of the present invention is on the rightmost side of each targeting ligand. It is understood that any RNAi and dsRNA molecules of the 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, the following are GLO-1 to Structure of GLO-16 and GLS-1 to GLS-16. GLO-1 GLS-1 GLO-2 GLS-2 GLO-3 GLS-3 GLO-4 GLS-4 GLO-5 GLS-5 GLO-6 GLS-6 GLO-7 GLS-7 GLO-8 GLS-8 GLO-9 GLS-9 GLO-10 GLS-10 GLO-11 GLS-11 GLO-12 GLS-12 GLO-13 GLS-13 GLO-14 GLS-14 GLO-15 GLS-15 GLO-16 GLS-16.

"Heterocycloalkyl" means a nonaromatic partially saturated or fully saturated ring (e.g., 3-10 or 3-7 membered heterocycloalkyl) having the specified number of ring atoms consisting of one or Composed of multiple heteroatoms (eg, 1, 2, 3, or 4 heteroatoms) selected from N, O, and S, and the remaining ring atoms are carbon. 5-membered heterocycloalkyl is a heterocycloalkyl group with 5 ring atoms. 6-membered heterocycloalkyl is a heterocycloalkyl group with 6 ring atoms. Heterocycloalkyl groups may be monocyclic or polycyclic (eg, bicyclic, tricyclic). Examples of heterocycloalkyl groups include oxanyl, aziridyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyridinyl, pyridazinyl, morpholinyl and Thiomorpholinyl. Additionally, one ring of a polycyclic heterocycloalkyl group can be aromatic (eg, aryl or heteroaryl) so long as the polycyclic heterocycloalkyl group is bonded to the parent structure via a nonaromatic carbon or nitrogen atom. For example, 1,2,3,4-tetrahydroquinolin-1-yl (where the moiety is bonded to the parent structure via a non-aromatic nitrogen atom) is considered a heterocycloalkyl group, while 1,2,3, 4-Tetrahydroquinolin-8-yl (where the moiety is bonded to the parent structure via an aromatic carbon atom) is not considered a heterocycloalkyl group. Lower heterocycloalkanes generally refer to C _3-6 monocyclic rings. Unless otherwise specified, lower heterocycloalkyl groups can generally be preferably fully saturated carbocyclic rings. In some embodiments of the invention, in vivo delivery may also be by beta-glucan delivery systems, 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 by reference. Incorporated herein. 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 invention, C3 dsRNA is delivered without 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 disorder in the subject, such as nephropathy .

It will be appreciated that in addition to certain delivery modalities described herein, other RNAi delivery modalities may be used in conjunction with embodiments of the C3 RNAi agents and therapeutic methods described herein, such as, but not limited to, those described herein and those used in the art. .

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

In some embodiments of the invention, the level of a C3 polypeptide in the cell, and thus its activity, is reduced by delivering (eg, introducing) a C3 dsRNA agent or C3 antisense polynucleotide agent into the cell. Targeting agents and methods can be used to facilitate delivery of C3 dsRNA agents or C3 antisense polynucleotide agents to specific cell types, cell subtypes, organs, spatial regions, and/or subcellular regions within cells 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 invention. In some embodiments, 2, 3, 4, or more independently selected C3 dsRNA agents are administered to the subject. In certain embodiments of the invention, a C3 dsRNA agent is administered to a subject in combination with one or more additional treatment regimens for treating a C3-related disease or disorder to treat a C3-related disease or disorder. Non-limiting examples of additional treatment regimens are administration of one or more C3 antisense polynucleotides of the invention, administration of non-C3 dsRNA therapeutics, and behavioral modification. Additional treatment regimens may be administered at one or more of the following times: before, simultaneously with, and after administration of the C3 dsRNA agent of the invention. It should be understood that "simultaneously" used in this article refers to within 5 minutes of zero time, within 10 minutes of zero time, within 30 minutes of zero time, within 45 minutes of zero time, and within 60 minutes of zero time, where "zero time" "Time" is the time at which a C3 dsRNA agent of the invention is administered to a subject. Non-limiting examples of non-C3 dsRNA therapeutics are: additional therapeutics 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 peptides Agents of inhibitors (e.g., simvastatin), thereby treating subjects with diseases that would benefit from reductions in C3 expression, or combinations of any of the foregoing, and formulated into combination agents for the treatment of paroxysmal epilepsy in adults and children Therapeutic agent for nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Non-limiting examples of behavior modification are: dietary programs, counseling, and exercise programs. These and other therapeutic agents and behavior modification agents are known in the art and may be used to treat C3 diseases or conditions in a subject, and may also be administered to a subject in combination with one or more C3 dsRNA agents of the invention to treat C3 Disease or illness. C3 dsRNA agents of the invention administered to a cell or subject to treat a C3-related disease or disorder can act in a synergistic manner with one or more other therapeutic agents or active ingredients, thereby adding one or more therapeutic agents or active ingredients effectiveness and/or increase the effectiveness of C3 dsRNA agents in treating C3-related diseases or conditions.

The treatment methods of the present invention include the administration of a C3 dsRNA agent, which may be used before the onset of and/or when a C3-related disease or disorder is present, including early, middle, late stages of the disease or disorder, and before any of these stages. and use at all times thereafter. The methods of the present invention may also treat subjects who have been previously treated for a C3-related disease or disorder with one or more other therapeutic agents and/or therapeutic active ingredients. Unsuccessful, minimally successful, and/or no longer successful in treating the subject's C3-related disease or condition. vector-encoded dsRNA

In certain embodiments of the invention, a carrier can be used to deliver C3 dsRNA agents into cells. C3 dsRNA reagent transcription units can be contained in DNA or RNA vectors. The preparation and use of such transgene-encoding vectors for delivering sequences into cells and/or subjects is well known in the art. Vectors that result in transient expression of C3 dsRNA, for example, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours, 1, 2, 3 can be used in the methods of the invention. , 4, 5, 6, 7, 8, 9, 10 or more weeks. The length of transient expression can be determined using conventional methods based on factors such as, but not limited to, the specific vector construct selected and the target cells and/or tissues. 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).

One or more single strands of the C3 dsRNA agent can be transcribed from a promoter on the 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 the cell using, for example, transfection or infection. In certain embodiments, each individual strand of the C3 dsRNA agent of the invention can be transcribed from a promoter contained on the same expression vector. In certain embodiments of the invention, the C3 dsRNA agent is expressed as inverted repeat polynucleotides linked by a linker polynucleotide sequence such that the C3 dsRNA agent has a stem-loop structure.

Non-limiting examples of RNA expression vectors are DNA plasmids or viral vectors. Expression vectors useful in embodiments of the invention may be compatible with eukaryotic cells. Eukaryotic 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, such as by intravenous or intramuscular administration, by administration to target cells removed from the subject and then reintroduced to the subject, or by any method that allows the introduction of the desired target cells. Proceed in other ways.

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 virus vector; (d) Herpes simplex virus vector; (e) SV 40 vector; (f) Polyomavirus vector; (g) Papillomavirus vector; (h) Picornavirus vector; (i) ) poxvirus vectors, such as orthopoxvirus vectors, such as vaccinia virus vectors or fowlpox virus vectors, such as canarypox or fowlpox virus vectors; (j) helper-dependent or gutless adenovirus vectors. Constructs for recombinant expression of C3 dsRNA agents may contain regulatory elements such as promoters, enhancers, etc., which may be selected to provide constitutive or regulated/inducible expression. Viral vector systems and the use of promoters and enhancers, etc. are routine in the art and can be used in conjunction with the methods and compositions described herein.

Certain embodiments of the invention include the use of viral vectors to deliver C3 dsRNA agents into cells. A number of 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 may be used in the methods of the invention are: AAV vectors, poxviruses such as vaccinia virus, modified Ankara virus (MVA), NYVAC, fowlpox such as fowlpox or canarypox virus.

Certain embodiments of the present invention include methods of delivering C3 dsRNA agents into cells using carriers, and such carriers may be in pharmaceutically acceptable carriers that may, but need not, include sustained release in which the gene delivery vector is embedded matrix. In some embodiments, vectors for delivering C3 dsRNA can be produced by recombinant cells, and pharmaceutical compositions of the invention can include one or more cells that produce C3 dsRNA delivery systems. Pharmaceutical compositions containing C3 dsRNA or ssRNA agents

Certain embodiments of the 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 C3 dsRNA agents or C3 antisense polynucleotide agents can be used in the methods of the invention to reduce C3 gene expression and C3 activity in cells, and can be used to treat C3-related diseases or disorders. 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 by parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for Compositions for intrathecal delivery, compositions formulated for direct delivery into the brain, and the like. The pharmaceutical compositions of the invention may be administered using one or more means to deliver the C3 dsRNA agent or C3 antisense polynucleotide agent into cells, e.g.: the surface (e.g., via a transdermal patch); the lungs, e.g. By inhalation or insufflation of powder or aerosol, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example by an implanted device; 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, such as directly to the liver, directly to the kidneys, and the like. It will be understood that "delivering a C3 dsRNA agent" or "delivering a C3 antisense polynucleotide agent" to a cell respectively includes delivering a C3 dsRNA agent or a C3 antisense polynucleotide agent, expressing the C3 dsRNA agent directly in the cell, and from The encoding vector is delivered to the cell expressing the C3 dsRNA agent, or any suitable means that causes the C3 dsRNA or C3 antisense polynucleotide agent to be present in the cell. The preparation and use of formulations and means for delivering inhibitory RNA are well known and routinely used in the art.

As used herein, a "pharmaceutical composition" includes a pharmacologically effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients, such as inert diluents, disintegrating agents, binders, lubricants, sweeteners, flavoring agents, and coloring agents. and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate and lactose, while cornstarch and alginic acid are suitable disintegrants. Binders may include starches and gelatin, while lubricants, if present, are usually magnesium stearate, stearic acid, or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. Agents included in the pharmaceutical formulations are described further below. As used herein, terms such as "pharmacologically effective amount,""therapeutically effective amount," and "effective amount" mean that the C3 dsRNA agent or C3 antisense polynucleotide agent of the invention produces the intended pharmacological, therapeutic, or preventive The amount of results. For example, a given clinical treatment is considered effective if it reduces a measurable parameter associated with the disease or disorder by at least 10%, then a therapeutically effective amount of a drug used to treat the disease or disorder is one that reduces that parameter by at least 10% required amount. 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 may comprise dsRNAi agents including, 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. In other embodiments, preferred dsRNAi agents in Table 2 include, for example, AV00375, AV00413, AV00416, or AV00429. effective amount

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

An 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 disorder is typically determined in a clinical trial in which a test population is compared with a control population in a blinded study Establish effective doses in populations. In some embodiments, an effective amount is an amount that results in a desired response, such as a reduction in C3-related disease or disorder in cells, tissues, and/or a subject suffering from the disease or disorder. Accordingly, an effective amount of a C3 dsRNA agent or a C3 antisense polynucleotide agent for treating a C3-related disease or condition treatable by reducing C3 polypeptide activity can be an amount that, when administered, reduces the amount of C3 polypeptide activity in the subject Reduce to less than the amount that would be present in the cell, tissue and/or subject without 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 Known as the "control" quantity. In some embodiments of the methods of the invention, the subject's control amount is the subject's pre-treatment amount; in other words, the subject's level before administration of the C3 agent can be the subject's control level and used to compare it to the subject's level after administration of the siRNA to the subject. C3 peptide activity and/or C3 gene expression levels are compared. In the case of treating a C3-related disease or disorder, the desired response may be to reduce or eliminate one or more symptoms of the disease or disorder in cells, tissues, and/or subjects. Reduction or elimination can be temporary or permanent. It will be appreciated that methods of determining C3 polypeptide activity, C3 gene expression, symptom assessment, clinical testing, and the like may be used to monitor the status of a C3-related disease or disorder. In some aspects of the invention, a desired response to treating a C3-related disease or disorder is to delay the onset of the disease or disorder or even prevent the onset of the disease or disorder.

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

Some embodiments of the invention include methods of determining the efficacy of a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention administered to a subject to treat a C3-related disease or disorder by assessing and/or monitoring the C3-related disease in the subject or one or more “physiological characteristics” of a condition. Non-limiting examples of physiological characteristics of C3-related diseases or conditions are that many patients also develop renal failure and an increased risk of thromboembolic events. Standard methods for determining this physiological profile are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, etc. The syndrome is defined by the presence of hemolysis, elevated liver enzymes, and low platelet count.

It will be appreciated that the amount of C3 dsRNA agent or C3 antisense polynucleotide agent administered to a subject may be modified based at least in part on such determination of the subject's determined disease and/or disorder state and/or physiological characteristics. The therapeutic amount can be altered by, for example, increasing or decreasing the C3-dsRNA agent or C3 by changing the composition with which the C3 dsRNA agent or C3 antisense polynucleotide agent is administered, by changing the route of administration, by changing the time of administration, etc. Amount of antisense polynucleotide reagent. 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 medical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of co-treatments, if any vary), the specific route of administration, and other factors within the health practitioner's knowledge and expertise. For example, the effective amount may depend on the desired level of C3 polypeptide activity and/or C3 gene expression effective in treating a C3-related disease or disorder. The skilled artisan can empirically determine the effective amount of a particular C3 dsRNA reagent or C3 antisense polynucleotide reagent for use in the methods of the invention without undue experimentation. In conjunction with the teachings provided herein, by selecting from among the various C3 dsRNA agents or C3 antisense polynucleotide agents of the invention and weighing, for example, potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred dosing Factors such as modality, can plan an effective preventive or curative treatment plan to effectively treat a specific subject. As used in embodiments of the invention, an effective amount of a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention may be an amount that produces a desired biological effect in the cell when contacted with the cell.

It will be appreciated that C3 gene silencing can be performed constitutively or by genome 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%. In some embodiments of the invention, by administering the C3 dsRNA agent 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%. Give medication

The C3 dsRNA agent and C3 antisense polynucleotide agent are delivered in a pharmaceutical composition in a dose sufficient to inhibit C3 gene expression. In certain embodiments of the invention, the dosage of the C3 dsRNA agent or C3 antisense polynucleotide agent is 0.01 to 200.0 mg per kilogram of recipient body weight per day, typically 1 to 50 mg/kg body weight per day, 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, inclusive. For example, each single administration of a C3 dsRNA agent or C3 antisense polynucleotide agent can be administered in a dosage ranging 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, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg /kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5 mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg /kg, 3.9 mg/kg, 4 mg/kg, 4.1 mg/kg, 4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg, 4.8 mg /kg, 4.9 mg/kg, 5 mg/kg, 5.1 mg/kg, 5.2 mg/kg, 5.3 mg/kg, 5.4 mg/kg, 5.5 mg/kg, 5.6 mg/kg, 5.7 mg/kg, 5.8 mg /kg, 5.9 mg/kg, 6 mg/kg, 6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7 mg/kg, 6.8 mg /kg, 6.9 mg/kg, 7 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg, 7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg /kg, 7.9 mg/kg, 8 mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.5 mg/kg, 8.6 mg/kg, 8.7 mg/kg, 8.8 mg /kg, 8.9 mg/kg, 9 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3 mg/kg, 9.4 mg/kg, 9.5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg /kg, 9.9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/ kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg , 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg to 50 mg/kg body weight.

Various factors may be considered in determining the dose and timing of delivery of the C3 dsRNA agents of the invention. The absolute amount of 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, the maximum dose may be used, ie, the highest safe dose based on sound medical judgment.

In some embodiments, methods of the invention may comprise administering to a subject 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a C3 dsRNA agent or C3 antisense polynucleotide Reagents. In some cases, a dose of a pharmaceutical compound (e.g., a C3 dsRNA-containing agent or a C3 antisense polynucleotide-containing agent) may be administered to the subject at least daily, every other day, weekly, every other week, monthly, etc., may be daily Administration may be administered once or more than once per day, for example 2, 3, 4, 5 or more times in a 24 hour period. The pharmaceutical composition of the present invention can be administered once daily; or the C3 dsRNA agent or C3 antisense polynucleotide agent can be administered in two, three or more sub-doses at appropriate intervals throughout the day, or even using Continuous infusion or delivered via controlled release formulation. In some embodiments of the methods of the invention, a pharmaceutical composition of the invention is administered to the subject once or more daily, once or more weekly, once or more monthly, or once or more annually.

In certain aspects, methods of the invention include 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 administration to a subject suffering from a C3-related disease or disorder Other drug therapies or treatment activities or combinations of regimens. Pharmaceutical compounds can be administered in the form of pharmaceutical compositions. Pharmaceutical compositions used in the methods of the present invention may 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 sufficient to reduce the activity of the C3 polypeptide at a weight or volume suitable for administration to the subject. The level in the unit that produces the desired response. The dosage of a pharmaceutical composition comprising a C3 dsRNA agent or a C3 antisense polynucleotide agent administered to a subject to reduce C3 protein activity may be selected based on different parameters, particularly based on the mode of administration used and the state of the subject. Other factors include the length of treatment required. If the subject's response at the initial dose is insufficient, a higher dose may be administered (or the dose may be effectively increased via a different, more local delivery route) as tolerated by the patient. treatment

As used herein, the term "prevent" or "prevent," when used to refer to a disease, disorder, or condition thereof that would benefit from reduced expression of the C3 gene, refers to the subject experiencing symptoms associated with such disease, disorder, or condition. The symptoms associated with such a disease, disorder or condition are, for example, symptoms associated with a disease or disorder caused by or associated with C3 activation, such as C3 glomerulopathy (C3G). The likelihood of developing C3 glomerulopathy (C3G) is reduced in situations such as when an individual has one or more risk factors for C3 glomerulopathy (C3G) relative to someone who has the same risk factors and does not have the same risk factors. Failure to develop the disease, condition, or condition, or a reduction in the development of symptoms associated with such disease, condition, or condition (e.g., clinically suffering from the disease, condition, or condition) in a person receiving treatment described herein Prevention is considered effective if it reduces the symptoms by at least approximately 10% on the scale), or delays the manifestation of symptoms (e.g., by days, weeks, months, or years).

C3-related diseases and disorders in which a reduction in the level and/or activity of a C3 polypeptide is effective in treating the disease or disorder can be treated using the methods and C3 dsRNA agents of the invention 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 therapeutic methods of the invention include, but are not limited to: C3 glomerulopathy (C3G), immune complex-mediated glomerulonephritis 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 other organ diseases known to be associated with complement dysfunction, such as age-related macular degeneration (AMD), rheumatoid arthritis (RA), antineutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegeneration disease. Such diseases and conditions may be referred to herein as "C3-related diseases and conditions" and "diseases and conditions caused and/or modulated by C3."

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 disorder. In some aspects of the invention, the subject is at risk of suffering from or developing a C3-related disease or disorder. A subject at risk of developing a C3-related disease or condition is a subject with an increased likelihood of developing a C3-related disease or condition compared to a control risk of developing a C3-related disease or condition. In some embodiments of the invention, the level of risk is statistically significant compared to a control level of risk. A subject at risk may include, for example: a subject who is or will be a subject with a pre-existing disease and/or genetic abnormality that renders the subject more susceptible to a C3-related disease or disorder than a control subject without the pre-existing disease or genetic abnormality; Subjects with a family and/or personal history of C3-related diseases or conditions; and subjects who have been previously treated 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 disorder may be a disease or genetic abnormality that, when present, has been previously determined to be associated with the development of a C3-related disease or disorder. Higher probability of correlation.

It will be appreciated that a C3 dsRNA agent or C3 antisense polynucleotide agent may be administered to a subject based on the individual subject's medical condition. For example, health care provided to a subject may evaluate C3 levels measured in a sample obtained from the subject and determine that it is desirable to reduce the subject's C3 levels by administering a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention. In one non-limiting example, a biological sample, such as a blood or serum sample, may be obtained from the subject and the subject's C3 level determined in the sample. Administering a C3 dsRNA reagent or C3 antisense polynucleotide reagent to a subject and obtaining a blood or serum sample from the subject after administration and using the sample to determine C3 levels and comparing the results to the subject's pre-dose (previous) sample Compare the determined results. A decrease in the subject's C3 levels in subsequent samples compared to pre-dose levels is indicative of the efficacy of the administered C3 dsRNA agent or C3 antisense polynucleotide agent in reducing the subject's C3 levels. In one non-limiting example, hemolysis may be considered a physiological characteristic of a C3-related disorder, even if the subject has not been diagnosed with a C3-related disorder, such as those disclosed herein. A healthcare provider can monitor changes in a subject's hemolysis as a measure of the efficacy of an administered C3 dsRNA agent or C3 antisense polynucleotide agent of the invention. In one non-limiting example, the 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., cytopenias.

Certain embodiments of the methods of the invention include modifying a treatment comprising administering to a subject an agent of the invention based at least in part on an assessment of a change in one or more physiological characteristics of a C3-related disease or disorder in the subject resulting from the treatment. dsRNA reagent or C3 antisense polynucleotide reagent. For example, in some embodiments of the invention, the effect of a dsRNA agent or C3 antisense polynucleotide agent of the invention administered to a subject can be determined and used to help modulate subsequent administration of a dsRNA agent or C3 antisense polynucleotide agent of the invention to the subject. Amount of sense polynucleotide agent. In one non-limiting example, a dsRNA agent or a C3 antisense polynucleotide agent of the invention is administered to a subject, and the subject's thrombus is determined after administration; and based at least in part on the determined levels, it is determined whether a higher amount of dsRNA is needed agent or C3 antisense polynucleotide agent to enhance the physiological effect of the administered agent, such as reducing or further reducing hemolysis in the subject. In another non-limiting example, a dsRNA agent or C3 antisense polynucleotide agent of the invention is administered to a subject, and the subject's hemolysis is determined following administration, and based at least in part on the determined levels, administration to the subject is expected to Lower amounts of dsRNA reagent or C3 antisense polynucleotide reagent.

Accordingly, some embodiments of the invention include assessing changes in one or more physiological characteristics resulting from prior treatment of a subject to adjust the amount of a dsRNA agent or C3 antisense polynucleotide agent of the invention subsequently administered to the subject. Some embodiments of the methods of the invention include 1, 2, 3, 4, 5, 6 or more determinations of physiological characteristics of a C3-related disease or disorder; assessing and/or monitoring administered C3 dsRNA agents or C3 of the invention the efficacy of the antisense polynucleotide agent; and optionally using the results of the assay to adjust one or more of the following: the effectiveness of the dsRNA agent or C3 antisense polynucleotide agent of the invention in treating a C3-related disease or disorder in a subject Dosage, dosing regimen, and/or dosing frequency. In some embodiments of the methods of the invention, the desired outcome of administering to a subject an effective amount of a dsRNA agent or C3 antisense polynucleotide agent of the invention is: C3 transcript levels, plasma Reduction in C3 levels and C3 gene expression.

As used herein, the terms "treat," "therapeutic," or "therapeutic" when used in reference to a C3-related disease or disorder may refer to preventive treatment, reducing the likelihood of a subject developing a C3-related disease or disorder, and may also refer to Treatment to eliminate or reduce the level of a C3-related disease or condition after a subject has developed a C3-related disease or condition, to prevent the C3-related disease or condition from becoming more severe, and/or to reduce C3 polypeptide activity in the subject in the absence of Slowing the progression of a C3-related disease or condition in a subject compared to the subject without the therapy.

Certain embodiments of the agents, compositions, and methods of the invention can be used to inhibit C3 gene expression. As used herein, with respect to the expression of a C3 gene, the terms "inhibit,""silence,""reduce,""down-regulate" and "knockdown" refer to altering the expression of the C3 gene, for example, by one or more of the following: respectively, A control level of RNA transcribed from a C3 gene, a control level of activity of expressed C3, or a control level of C3 translated from mRNA, when a cell, cell population, tissue, organ or subject is treated with a C3 dsRNA agent of the invention or a C3 The level of RNA transcribed from the gene, the activity level of C3 expressed, and the C3 translated from the mRNA in the cell, cell population, tissue, organ, or subject upon exposure to (e.g., treatment with) an antisense polynucleotide agent Decreased levels of peptides, proteins, or protein subunits. In some embodiments, the control level is the level in a cell, tissue, organ or subject that has not been exposed to (eg, treated with) a C3 dsRNA agent or a C3 antisense polynucleotide agent. Application method

A variety of routes of administration of C3 dsRNA agents or C3 antisense polynucleotide agents may be used in the methods of the invention. The selection of a particular delivery mode 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 may be carried out using any mode of administration that is medically acceptable, meaning any mode that produces effective therapeutic levels for a C3-related disease or condition without causing clinically unacceptable side effects. In some embodiments of the invention, a C3 dsRNA agent or C3 antisense polynucleotide agent can be administered by oral, enteral, mucosal, subcutaneous, and/or parenteral routes. 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 gastronasal tube), transdermal, vaginal, rectal, sublingual, and inhalation. Delivery routes of the present invention may include intrathecal, intraventricular, or intracranial. In some embodiments of the invention, a C3 dsRNA agent or C3 antisense polynucleotide agent can be placed in a sustained release matrix and administered by placing the matrix in a subject. In some aspects of the invention, C3 dsRNA agents or C3 antisense polynucleotide agents can be delivered to cells of a subject using nanoparticles coated with a delivery agent that targets specific cells or organelles. A variety of delivery modes, methods, and reagents are known in the art. Non-limiting examples of delivery methods and delivery agents are provided elsewhere herein. In some aspects of the invention, the term "delivery" with respect to a C3 dsRNA agent or a C3 antisense polynucleotide agent may refer to the administration of one or more "naked" C3 dsRNA agents or C3 antisense polynucleotides to a cell or subject Acid sequence. In certain aspects of the invention, "delivery" means 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, or delivering a C3 dsRNA agent or C3 antisense polynucleotide encoding a C3 dsRNA agent to a subject. Carriers of polynucleotide agents are delivered to cells and/or subjects and the like. Delivery of a C3 dsRNA agent or C3 antisense polynucleotide agent using transfection may include administration of a vector to the cell and/or subject.

In some methods of the invention, one or more C3 dsRNA reagents or C3 antisense polynucleotide reagents may be administered in the form of a preparation or in a pharmaceutically acceptable solution, which may generally contain a pharmaceutically acceptable solution. Acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. In some embodiments of the invention, a C3 dsRNA agent or C3 antisense polynucleotide agent can be formulated with another therapeutic agent for simultaneous administration. 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, pharmaceutical compositions comprise a C3 dsRNA agent or C3 antisense polynucleotide agent and optionally a 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 nontoxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (e.g., the ability of a C3 dsRNA agent or a C3 antisense polynucleotide agent to inhibit C3 gene expression in a cell or subject). Various 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 invention.

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, and other carriers are known to those skilled in the art. Such preparations may generally 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, which 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, propionic acid. Diacid, succinic acid, etc. Furthermore, pharmaceutically acceptable salts may be prepared as alkali metal salts or alkaline earth metal salts, such as sodium, potassium or calcium salts.

Some embodiments of the methods of the invention include administering one or more C3 dsRNA agents or C3 antisense polynucleotide agents directly to the tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which a C3-related disease or disorder exists or may occur, non-limiting examples of which are the liver or kidneys. Direct tissue drug delivery 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 to a subject one or more C3 dsRNA agents. 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 may be administered subcutaneously, and one or more additional administrations may be oral and/or systemic.

For embodiments of the invention in which it is desired to administer the C3 dsRNA agent or C3 antisense polynucleotide agent systemically, the C3 dsRNA agent or C3 antisense polynucleotide agent may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. . Injectable preparations may be presented in unit dosage form such as ampoules or multi-dose containers, with or without added preservatives. C3 dsRNA agent preparations (also referred to as pharmaceutical compositions) may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Formulations 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, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose solution, dextrose 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), and the like. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents, inert gases, etc. may also be present. Other forms of administration, such as intravenous administration, will result in lower doses. If the subject's response at the initial dose is inadequate, a higher dose may be administered (or the dose may be effectively increased via a different, more local delivery route) as tolerated by the patient. Multiple daily doses may 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 reductions in C3 protein activity.

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

Both non-biodegradable and biodegradable polymeric matrices can be used in the methods of the 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. Matrix polymers can be natural or synthetic polymers. The polymer can be selected based on the period of time for which release is desired, typically on the order of a few hours to a year or more. Typically, releases for periods ranging from a few hours to three to twelve months are available. The polymer is optionally in the form of a hydrogel, which can absorb up to about 90% of its weight in water, and is optionally also cross-linked with multivalent ions or other polymers.

Generally, C3 dsRNA agents or C3 antisense polynucleotide agents in some embodiments of the invention may be delivered by diffusion or by degradation of the polymeric matrix using biodegradable implants. Exemplary synthetic polymers for this purpose are well known in the art. Biodegradable polymers and non-biodegradable polymers can be used to deliver C3 dsRNA agents or C3 antisense polynucleotide agents using methods known in the art. Bioadhesive polymers such as bioerodible hydrogels (H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587) can also be used to deliver C3 dsRNA reagents or C3 antisense polynucleotide reagents for therapeutic purposes C3 related diseases or conditions. Other suitable delivery systems may include timed release, delayed release, or sustained release delivery systems. Such systems may avoid repeated administration of C3 dsRNA reagents or C3 antisense polynucleotide agents, thereby improving convenience for subjects 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 are available, some of which are also suitable for implantation.

The use of long-term sustained release implants may be suitable for 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 for at least up to 10 days, 20 days, 30 days, 60 days, 90 days, six months, one year, or longer dsRNA reagent or C3 antisense polynucleotide reagent. 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.

Therapeutic formulation of C3 dsRNA agents or C3 antisense polynucleotide agents can be achieved by combining the molecule or compound with the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer [Remington's Pharmaceutical Sciences 21st edition, ( 2006)] are mixed for storage in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations employed and include buffering agents such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine ; Preservatives (e.g. stearyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; parabens, e.g. Methyl or propyl hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, e.g. Serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; simple sugars, Disaccharides and other carbohydrates, including glucose, mannose or dextrin; 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 non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). cells, objects and controls

The methods of the 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 vertebrate mammal, including but not limited to dogs, cats, horses, cattle, goats, mice, rats, and primates, such as monkeys. Accordingly, the present invention may be used to treat C3-related diseases or conditions in humans and non-human subjects. In some aspects of the invention, the subject may be a farm animal, a zoo animal, a domesticated animal, or a non-domesticated animal, and the methods of the invention may be used in veterinary preventive and therapeutic regimens. In some embodiments of the invention, the subject is a human and the methods of the invention can be used in human prophylactic and therapeutic regimens.

Non-limiting examples of subjects to which the present invention may be applied are subjects diagnosed with, suspected of having, or at risk of suffering from a disease or condition associated with: higher than expected C3 expression and/or activity, 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 may be applied to subjects who, at the time of treatment, have been diagnosed with the disease or disorder, are associated with higher than expected C3 expression and/or activity, or are thought to be suffering from or developing conditions associated with higher than expected C3 expression and/or activity. Desirably, the subject is at risk for a disease or condition associated with C3 expression and/or activity. 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 desirable levels of C3 expression and/or activity The relevant disease or condition is a chronic disease or condition.

In a non-limiting example, C3 dsRNA agents of the invention are administered to patients diagnosed with 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 methods of the present invention may be applied to subjects who, at the time of treatment, have been diagnosed as having the disease or condition, or who are considered to be at risk of developing or developing the disease or condition.

In another non-limiting example, a C3 dsRNA agent of the invention is administered to treat a disease or disorder caused by or associated with activation of C3, or a disease or disorder whose symptoms or progression are in response to inactivation of C3. The term "C3-related disease" includes diseases, disorders or conditions that benefit from reduced expression of C3. These disorders are often associated with hemolysis, elevated liver enzymes, and low platelet counts as physiological features of the disease. 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, the C3-related disease includes paroxysmal nocturnal hemoglobinuria (PNH).

In another non-limiting example, a C3 dsRNA agent of the invention is administered to treat a molecule for inhibiting C3 gene expression in neural cells, such as neural cells in a subject, such as a mammal, such as a patient with C3 People with related neurodegenerative diseases, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), schizophrenia, Parkinson's disease (PD), and prion diseases, such as Creutzfeldt-Jakob disease ( CJD).

Cells to which the method of the present invention can be applied include in vitro, in vivo, and ex vivo cells. The cells may be in a subject, in culture and/or in suspension, or in any other suitable state or condition. The cells to which the method of the present invention can be applied may be: liver cells, hepatocytes, heart cells, pancreatic cells, cardiovascular cells, kidney cells or other types of vertebrate cells, including human and non-human mammalian cells. animal cells. In certain aspects of the invention, cells to which the methods of the invention can be applied are healthy normal cells that are not known to be disease cells. In certain embodiments of the invention, the methods and compositions of the invention are applied to cells of the liver, hepatocytes, heart cells, pancreatic cells, cardiovascular cells, and/or renal cells. In certain aspects of the invention, control cells are normal cells, but it should be understood that cells having a disease or disorder may also be used as control cells in certain circumstances, such as when comparing treated cells having a disease or disorder to cells having a disease or disorder. In the case of other diseases that result from untreated cells.

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 many forms. It can be a single cutoff value, such as the median or mean. It may be established based on comparative groups, for example in a group with normal levels of C3 polypeptide and/or C3 polypeptide activity and in a group with increased levels of C3 polypeptide and/or C3 polypeptide activity. Another non-limiting example of a comparison group may be a population with one or more symptoms or diagnosis of a C3-related disease or disorder versus a population without one or more symptoms or diagnosis of the disease or disorder; to which the present invention has been administered The subject group treated with the siRNA of the present invention and the subject group to which the siRNA treatment of the present invention was not administered. Typically, controls can be based on apparently healthy normal individuals or apparently healthy cells in an appropriate age group. It will be understood that, in addition to predetermined values, a control according to the present invention may be a material sample tested in parallel with the experimental material. Examples include samples from control populations or control samples generated through manufacturing for parallel testing with experimental samples. In some embodiments of the invention, controls may include cells or subjects that have not been contacted or treated with a C3 dsRNA agent of the invention, in which case control levels of C3 polypeptides and/or C3 polypeptide activity may be compared to those of the invention. The level of C3 polypeptide and/or C3 polypeptide activity in a cell or subject contacted by an inventive C3 dsRNA agent or C3 antisense polynucleotide agent.

In some embodiments of the invention, the control level may be a C3 polypeptide level determined for a subject to which C3 polypeptide levels determined at different times for the same subject are compared. In one non-limiting example, C3 levels are determined in biological samples obtained from subjects who have not received C3 treatment of the present invention. In some embodiments, the biological sample is a serum sample. The C3 polypeptide level measured in a sample obtained from a subject may serve as a baseline or control value for the subject. After one or more administrations of a C3 dsRNA agent to a subject in a treatment method of the 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 Compare to subject's control/baseline levels. Such comparisons may be used to assess the onset, progression, or regression of C3-related diseases or conditions in a subject. For example, a C3 polypeptide level in a baseline sample obtained from a subject that is greater than the level obtained from the same subject after administration of a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention to the subject is indicative of regression of a C3-related disease or condition and Indicates the efficacy of an administered C3 dsRNA agent of the invention in treating a C3-related disease or disorder.

In certain aspects of the invention, one or more of the C3 polypeptide and/or C3 polypeptide activity levels determined for a subject can serve as a control value and be used to later compare C3 polypeptide and/or C3 activity in the same subject. levels, thereby allowing assessment of changes in "baseline" C3 polypeptide activity in subjects. Therefore, where the initial level is used as a control level for the subject, the initial C3 polypeptide level and/or the initial C3 polypeptide activity level can be used to demonstrate and/or determine the ability of the methods and compounds of the invention to reduce the Levels of C3 polypeptide and/or C3 polypeptide activity.

Using the methods of the invention, a C3 dsRNA agent and/or C3 antisense polynucleotide agent of the 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 administration and treatment of the invention may be assessed as compared to pre-dose levels of C3 polypeptide in serum samples obtained from the subject at previous time points, or to non-exposure control levels (e.g., C3 polypeptide levels in control serum samples) Compared to, when administered and after treatment, the level of C3 polypeptide in serum samples obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 90%, 95% or more. It will be appreciated that both the level of C3 polypeptide and the level of C3 polypeptide activity are related to the level of C3 gene expression. Certain embodiments of the methods of the invention include administering to a subject a C3 dsRNA and/or C3 antisense agent of the 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 (also referred to herein as treatment) of a cell with an siRNA agent of the invention results in an inhibition of C3 gene expression in the cell by 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%, or about 100%, for example, to below the assay detection level.

Some embodiments of the invention include determining the presence, absence and/or amount (also referred to herein as levels) of a C3 polypeptide in one or more biological samples obtained from one or more subjects. This assay can be used to assess the efficacy of the treatment methods of the invention. For example, the methods and compositions of the invention can be used to determine the levels of C3 polypeptides in biological samples obtained from subjects previously treated with administration of a C3 dsRNA agent and/or C3 antisense agent of the invention. Compared to pre-dose levels of C3 polypeptides in serum samples obtained from the subject at previous time points, or compared to non-exposure control levels (e.g., C3 polypeptide levels in control serum samples) obtained from the subject after administration and treatment The level of C3 polypeptide in the serum sample is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more More indicates the level of efficacy of the treatment given to the subject.

In some embodiments of the present invention, the physiological characteristics of a C3-related disease or disorder determined for a subject can be used as a control result, and the determination of the physiological characteristics of the same subject at different times is compared with the control results. In one non-limiting example, pathological characteristic hemolysis is measured from subjects who have never been administered a C3 treatment of the invention, which can be used as a baseline or control value for the subject. After one or more administrations of a C3 dsRNA agent to a subject in a treatment method of the invention, the blood cells are compared to the subject's control/baseline levels, respectively. Such comparisons may be used to assess the onset, progression, or regression of C3-related diseases or conditions in a subject. For example, baseline blood cells obtained from a subject that are higher than thrombi determined from the same subject after administration of a C3 dsRNA agent or C3 antisense polynucleotide agent of the invention to the subject would indicate regression of the C3-related disease or disorder and would indicate that the administered Efficacy of the invented C3 dsRNA reagents in treating C3 related diseases or conditions.

Some embodiments of the present invention include determining the presence, absence, and/or changes in physiological characteristics of a C3-related disease or disorder using methods such as, but not limited to: (1) measuring a subject's blood cells; (2) assessing a patient's blood cells from a patient or more Physiological characterization of one or more biological samples obtained from multiple subjects; (3) or physical examination of the subject. This assay can be used to assess the efficacy of the treatment methods of the invention. pill box

Kits containing one or more C3 dsRNA reagents and/or C3 antisense polynucleotide reagents and instructions for their use in the methods of the invention are also within the scope of the invention. Kits of the invention may include one or more of a C3 dsRNA agent, a C3 sense polynucleotide, and a C3 antisense polynucleotide agent useful in treating C3-related diseases or disorders. Kits containing one or more C3 dsRNA reagents, C3 sense polynucleotides, and C3 antisense polynucleotide reagents can be prepared for use in the treatment methods of the invention. The components of the kit of the invention may be packaged in an aqueous medium or in lyophilized form. Kits of the present invention may comprise a carrier divided to receive therein one or more container means or a series of container means (eg test tubes, vials, flasks, bottles, syringes, etc.). The first container device or series of container devices may contain one or more compounds, such as a C3 dsRNA agent and/or a C3 sense or antisense polynucleotide agent. The second container device or series of container devices may contain a targeting agent, labeling agent, delivery agent, etc., which may serve as a component of the C3 dsRNA agent and/or C3 antisense polynucleotide administered in embodiments of the treatment methods of the invention. Partially included.

The kit of the present invention may also contain instructions. Instructions are usually in written form and will provide guidance for performing the treatment embodied by the pill box and making decisions based on that treatment.

The following examples are provided to illustrate specific examples of the practice of the 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 is applicable to a variety of compositions and methods. Example

Example 1. Synthesis of RNAi reagents

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

siRNA sense and antisense strand sequences are synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. Oligonucleotide chain growth is achieved through a 4-step cycle: deprotection, condensation, capping, and an oxidation or sulfation step for the addition of each nucleotide. The synthesis was performed on a solid support made of controlled porous glass (CPG, 1000 Å). Monomeric phosphoramidite was 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 siRNA for in vitro screening (Table 2), synthesis was performed at a 2 μmol scale; for siRNA for in vivo testing (Table 3), synthesis was performed at a scale of 5 μmol or larger. In the case where a GalNAc ligand (GLO-0 as a non-limiting example) is attached to the 3'-end of the sense strand, a CPG solid support to which the GalNAc ligand is attached is used. In the case where a GalNAc ligand (GLPA1, GLS2 or GLS-15 as a non-limiting example) is attached to the 5'-end of the sense strand, a GalNAc phosphoramidite (GLPA1, GLPA2 or GLPA15 as a non-limiting example) ) for the final coupling reaction. Trichloroacetic acid (TCA) in 3% dichloromethane was used for deprotection of the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole was used as activator. I in THF/Py/H ₂ O and phenylacetyl disulfide (PADS) in pyridine _/ MeCN were used for the oxidation and sulfidation reactions, respectively. After the final solid-phase synthesis step, the solid support-bound oligomers were cleaved and protecting groups were 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-chain 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). Convert the purified single-stranded oligonucleotide product from IP-RP-HPLC to its sodium salt by dissolving it in 1.0 M NaOAc and precipitating it by adding ice-cold EtOH. Annealing of sense and antisense strand oligonucleotides is performed by equimolar complementation in water to form a double-stranded siRNA product, which is lyophilized to provide a fluffy white solid.

Example 2. Preparation of Intermediate-A and Intermediate-B

Intermediate-A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl triflate (TMSOTf) in dichloromethane (DCM) as shown in Scheme 1 below. Glycosylation with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol then provided compound II. The Cbz protecting group is removed by hydrogenation to provide Intermediate-A as the trifluoroacetate (TFA) salt. Intermediate B was synthesized based on the same protocol except using Cbz protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol as starting material. Intermediate-A Intermediate-B plan 1

To a solution of compound I (20.0 g, 51.4 mmol) in 100 mL 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), dried with 4 Å powdered molecular sieve (10 g), and added dropwise to the above reaction solution at 0 °C under _N2 atmosphere. The resulting reaction mixture was stirred at 25 °C for 16 h under _N2 atmosphere. The reaction mixture was filtered and washed with saturated _NaHCO3 (200 mL), water (200 mL) and saturated brine (200 mL). The organic layer was dried over _{anhydrous Na2SO4} , filtered and concentrated under reduced pressure to obtain the crude product, which was triturated with 2-methyltetrahydrofuran/heptane (5/3, v/v, 1.80L) for 2 hours. The resulting mixture was filtered and dried to obtain compound II as a white solid (15.0 g, yield 50.3%).

10% Pd/C (1.50 g) was carefully added to a dry and argon-purged hydrogenation flask, followed by 10 mL of tetrahydrofuran (THF), followed by 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 a _H2 (45 psi) atmosphere. Thin layer chromatography (TLC, solvent: DCM:MeOH = 10:1) showed complete consumption of compound II. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. This process was repeated three times to obtain Intermediate-A as a foamy white solid (14.0 g, yield 96.5%). 1H NMR (400 MHz DMSO-d6): δ ppm 7.90 (d, J = 9.29 Hz, 1 H), 7.78 (br s, 3 H), 5.23 (d, J = 3.26 Hz, 1 H), 4.98 (dd , J = 11.29, 3.26 Hz, 1 H), 4.56 (d, J = 8.53 Hz, 1 H), 3.98 - 4.07 (m, 3 H), 3.79 - 3.93 (m, 2 H), 3.55 - 3.66 (m , 5 H), 2.98 (br d, J = 4.77 Hz, 2 H), 2.11 (s, 3 H), 2.00 (s, 3 H), 1.90 (s, 3 H), 1.76 (s, 3 H) .

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

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

Prepare GLPAl and GLPA2 according to Scheme 2 below. Starting from benzyl-protected propane-1,3-diamine, alkylation with tert-butyl 2-bromoacetate affords triester compound I. Removal of the benzyl protecting group by hydrogenation affords secondary amine compound II. Coupling of amide with 6-hydroxycaproic acid affords compound III. The tert-butyl protecting group is then removed upon treatment with HCl in dioxane to yield triacid compound IV. Amide coupling between triacid compound IV and Intermediate-A or Intermediate-B is performed to provide compound Va or Vb. Phosphoramidite GLPA1 or GLPA2 is synthesized by phosphorylation of compound Va or Vb with 2-cyanoethyl N,N-diisopropyl chloride phosphoramidite and a catalytic amount of 1H-tetrazole. Scenario 2

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); then add diisopropylethylamine (DIEA, 23.61g, 182mmol) dropwise. The resulting reaction mixture was stirred at 25-30°C for 16 hours. LCMS showed complete consumption of N-benzyl-1,3-propanediamine. The reaction mixture was diluted with H ₂ O (500 mL) and extracted with EtOAc (500 mL × 2). The combined organic matter was washed with saturated brine (1L), dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure to obtain a crude product; purified by silica gel column chromatography (gradient: petroleum ether: ethyl acetate 20:1 to 5: 1). Compound I was obtained as colorless oil (12.1 g, yield 78.4%). 1H NMR (400 MHz, CDCl3): δ ppm 7.26 - 7.40 (m, 5 H), 3.79 (s, 2 H), 3.43 (s, 4 H), 3.21 (s, 2 H), 2.72 (dt, J = 16.9, 7.34 Hz, 4 H), 1.70 (quin, J = 7.2 Hz, 2 H), 1.44 - 1.50 (m, 27 H).

The dry hydrogenation bottle was purged three times with argon. Pd/C (200 mg, 10%) was added, followed by MeOH (5 mL), then compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with _H2 . This process is repeated 3 times. The mixture was stirred at 25°C for 12 hours under an atmosphere of _H2 (15 psi). LCMS showed that Compound I was completely consumed. The reaction mixture was filtered under reduced pressure under _N2 atmosphere. The filtrate was concentrated under reduced pressure to obtain compound II (655 mg, yield 79.7%) as a yellow oil, which was used in the next step without further purification. 1H NMR (400 MHz, CDCl3): δ ppm 3.44 (s, 4 H), 3.31 (s, 2 H), 2.78 (t, J = 7.1 Hz, 2 H), 2.68 (t, J = 6.9 Hz, 2 H), 1.88 (br s, 1 H), 1.69 (quin, J = 7.03 Hz, 2 H), 1.44 - 1.50 (s, 27 H).

Compound II (655mg, 1.57mmol), 6-hydroxycaproic acid (249mg, 1.89mmol), DIEA (1.02g, 7.86mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide A mixture of hydrochloride (EDCI, 904 mg, 4.72 mmol) and 1-hydroxybenzotriazole (HOBt, 637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged 3 times with N2; then under _N2 atmosphere Stir at 25°C for 3 hours. LCMS indicated the desired product. The reaction mixture was diluted with H ₂ O (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; this was passed through silica gel column chromatography (gradient: petroleum ether: ethyl acetate from 5:1 to 1:1 ) was purified to obtain compound III (650 mg, yield 77.8%) as yellow oil. 1H NMR (400 MHz, CDCl3): δ ppm 3.90 - 3.95 (s, 2 H), 3.63 (t, J = 6.40 Hz, 2 H), 3.38 - 3.45 (m, 6 H), 2.72 (t, J = 6.65 Hz, 2 H), 2.40 (t, J = 7.28 Hz, 2 H), 1.55 - 1.75 (m, 8 H), 1.44 (s, 27 H). Calculated mass spectrum C27H50N2O8: 530.36; measured: 531.3 (M+ H ⁺ ).

A mixture of compound III (5.5 g, 10.3 mmol) in HCl/dioxane (2M, 55 mL) was stirred at 25 °C for 3 h. LCMS showed complete consumption of compound III. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to obtain crude product. Dissolve this in CH3CN (50 mL) and remove volatiles in vacuo. This process was repeated three times to obtain compound IV as a white solid (2.05 g, yield 54.5%). ¹ H NMR (400 MHz, D2O): δ ppm 4.21 (s, 1 H), 4.07 (d, J = 4.5 Hz, 4 H), 3.99 (s, 1 H), 3.45 - 3.52 (m, 3 H) , 3.42 (t, J = 6.5 Hz, 1 H), 3.32 - 3.38 (m, 1 H), 3.24 - 3.31 (m, 1 H), 2.37 (t, J = 7.4 Hz, 1 H), 2.24 (t , J = 7.4 Hz, 1 H), 1.99 (dt, J = 15.5, 7.53 Hz, 1 H), 1.85 - 1.94 (m, 1 H), 1.85 - 1.94 (m, 1 H), 1.39 - 1.56 (m , 4 H), 1.19 - 1.31 (m, 2 H).

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 in DMF (10 mL) (496 mg, 3.67 mmol) was degassed and purged 3 times with _N2 , and then the mixture was stirred at 25 °C under _N2 atmosphere for 3 h. LCMS indicated the desired product. The reaction mixture was quenched by adding H ₂ O (10 mL) and extracted with DCM (10 mL × 2). The combined organics were extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with saturated _NaHCO solution and re-extracted with DCM (10 mL x 2). The organics were dried over sodium sulfate, filtered and concentrated under reduced pressure to give compound Va as a white solid (570 mg, 0.281 mmol, 26.8% yield). ¹ H NMR: (400 MHz, CDCl3) ppm δ 7.84 - 8.12 (m, 3 H), 6.85 - 7.15 (m, 2 H), 6.66 - 6.81 (m, 1 H), 5.36 (br d, J = 2.7 Hz, 3 H), 5.11 - 5.27 (m, 3 H), 4.63 - 4.85 (m, 3 H), 3.90 - 4.25 (m, 18 H), 3.37 - 3.75 (m, 28 H), 3.15 - 3.28 ( m, 4 H), 2.64 (br d, J = 6.53 Hz, 2 H), 2.30 - 2.46 (m, 2 H), 2.13 - 2.18 (m, 9 H), 2.05 (s, 9 H), 1.94 - 2.03 (m, 18 H), 1.68 (br s, 2 H), 1.45 (br s, 2 H), 1.12 (br t, J = 7.0 Hz, 2 H).

To a solution of compound Va (260 mg, 0.161 mmol) in anhydrous DCM (5 mL) was added tetrazole diisopropylammonium (30.3 mg, 0.177 mmol); then ₃ -bis(diisopropylammonium) was added dropwise at ambient temperature and N Isopropylamino)phosphonyloxypropionitrile (194 mg, 0.645 mmol). 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 NaHCO ₃ (1:1, 5 mL) solution at 0°C. After stirring for 1 minute, DCM (5 mL) was added. Stratification occurs. The organic layer was washed with brine/saturated _aqueous NaHCO solution (1 _: 1, 5 mL), dried over _Na2SO4 , filtered and concentrated to a volume of approximately 1 mL. The remaining solution was added dropwise to 20 mL of methyl tert-butyl ether (MTBE) while stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged and the solids were collected. The solid was redissolved in 1 mL DCM and precipitated by adding MTBE (20 mL). The solid was separated again by centrifugation. The collected solid was dissolved in anhydrous CH3CN. Remove volatiles. 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 (400 MHz, CDCl3): ppm δ 7.71 - 8.06 (m, 2 H), 6.60 - 7.06 (m, 3 H), 5.37 (br d, J = 3.0 Hz, 3 H), 5.18 - 5.32 (m , 3 H), 4.70 - 4.86 (m, 3 H), 3.92 - 4.25 (m, 18 H), 3.42 - 3.85 (m, 30 H), 3.25 (m, 4 H), 2.59 - 2.75 (m, 4 H), 2.27 - 2.44 (m, 2 H), 2.15 - 2.20 (s, 9 H) 2.07 (s, 9 H), 1.96 - 2.03 (m, 18 H), 1.65 (br s, 4 H), 1.44 (br d, J = 7.28 Hz, 2 H), 1.14 - 1.24 (m, 12 H). 31P NMR (CDCl3): ppm δ 147.15.

The GalNAc ligand phosphoramidite compound GLPA2 was synthesized using the same procedure except that Intermediate-B was used. 1H NMR (400 MHz, CDCl3): ppm δ 7.94 - 8.18 (m, 1 H), 7.69 (br s, 1 H), 6.66 - 7.10 (m, 3 H), 5.35 (d, J = 3.5 Hz, 3 H), 5.07 - 5.25 (m, 3 H), 4.76 - 4.86 (m, 3 H), 4.01 - 4.31 (m, 10 H), 3.91 - 4.01 (m, 8 H), 3.74 - 3.86 (m, 4 H), 3.52 - 3.71 (m, 30 H), 3.42 - 3.50 (m, 6 H), 3.15 - 3.25 (m, 4 H), 2.52 - 2.70 (m, 4 H), 2.22 - 2.45 (m, 2 H), 2.15 - 2.22 (s, 9 H), 2.06 (s, 9 H), 1.95 - 2.03 (m , 18 H), 1.77 (br s, 2 H), 1.58 - 1.66 (m, 4 H), 1.40 (m, 2 H), 1.08 - 1.24 (m, 12 H). 31P NMR (CDCl3): ppm δ 147.12.

Prepare GLPA15 following Scheme 3 below.

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.), and then Cbz-Cl was added dropwise (169 g, 990 mmol, 1.50 eq.). The reaction solution was stirred at 25°C for 2 hours, and LCMS showed that compound II was completely converted. The reaction solution was washed with a saturated solution of NaHCO3 (800 mL) and saturated brine (500 mL) in sequence, and the organic phase was dried over anhydrous Na ₂ SO ₄ . After filtering to remove the desiccant, the filtrate was concentrated to dryness. The crude product was subjected to column chromatography (SiO ₂ , PE/EA=100/1 to 5/1) to obtain colorless oily compound III-1 (290 g, 527 mmol, yield 75.7%). ¹ H NMR (400 MHz in DMSO-d6): δ ppm 7.23 - 7.40 (m, 5 H), 5.00 - 5.12 (m, 2 H), 3.86 - 3.95 (m, 2 H), 3.23 - 3.39 (m, 6 H), 2.55 - 2.67 (m, 2 H), 1.56 - 1.64 (m, 2 H), 1.31 - 1.46 (m, 27 H). MS (ESI) [M+H] ⁺ m/z: 551.6.

HCOOH (2.9 L) was added to compound III-1 (145 g, 263 mmol, 1.00 eq ), and the solution was stirred at 60 °C for 12 hours. LCMS showed that compound III-1 was completely converted. Add 1.5 L toluene and 1.5 L acetonitrile to the reaction solution, concentrate under reduced pressure to about 500 mL, then add toluene/acetonitrile (1:1, ~750 mL) and concentrate to about 500 mL, then add acetonitrile (~1000 mL) And concentrated to dryness, the crude product was pulverized with 700 mL acetonitrile at 60°C for 2 hours and filtered. The solid was collected and dried to obtain compound IV-1 as a white solid (105 g, quantitative). 1H NMR (400 MHz in DMSO-d6): δ ppm 7.26 - 7.40 (m, 5 H), 5.02 - 5.10 (m, 2 H), 3.89 - 4.00 (m, 2 H), 3.36 - 3.45 (m, 4 H), 3.24 - 3.34 (m, 2 H), 2.59 - 2.72 (m, 2 H), 1.40 (s, 2 H). MS (ESI) [M+H] ⁺ m/z: 383.0.

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

Inert the 2 L hydrogenation kettle with argon and carefully add dry Pd/C (9 g), add MeOH (50 mL) to moisten the Pd/C, and then slowly add 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). The mixture was replaced with hydrogen atmosphere by degassing/adding H ₂ three times, and stirred at 25°C for 10 hours. LCMS showed that compound V-1 was completely converted. Pd/C was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain compound VI-1 (80 g, yield 90.2%). ¹ H NMR (400 MHz in DMSO-d6): δ ppm 9.12 (br s, 2 H), 8.50 (br t, J=5.19 Hz, 1 H), 8.10 (br t, J=5.50 Hz, 2 H), 7.85 - 7.91 (m, 3 H), 5.22 (d, J=3.25 Hz, 3 H), 4.95 - 5.01 (m, 3 H), 4.52 - 4.58 (m, 3 H), 4.03 (s, 9 H), 3.84 - 3.93 (m, 3 H), 3.75 - 3.83 (m, 3 H), 3.39 - 3.61 (m, 16 H), 3.23 - 3.32 (m, 6 H), 3.15 - 3.18 (m, 3 H), 2.97 - 3.05 (m, 2 H), 2.54 - 2.61 (m, 2 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 - 1.80 (m, 9 H), 1.70 - 1.76 (m, 2 H) . MS (ESI) [M+H] ⁺ m/z: 749.3.

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, and LCMS showed that compound VI-1 was completely converted into compound VII. 4-Hydroxypyridine (42.4 g, 420 mmol, 2.50 eq. ) and TBTU (107 g, 335 mmol, 2.00 eq. ) were added to the reaction solution, and stirring was continued at 25°C for 1 hour. LCMS showed complete conversion of compound VII. The reaction was quenched by slowly adding 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 methylene chloride and added dropwise to methyl tert-butyl ether (7.5 L). A translucent white precipitate gradually formed during the dropping process. The precipitate was filtered under vacuum, and the solid was collected and dried under vacuum to obtain compound VIII as a white solid (207 g, yield 72.8%). ¹ H NMR (400 MHz in DMSO-d6): δ ppm 8.05 (br d, J=2.00 Hz, 2 H), 7.82 (br d, J=7.38 Hz, 3 H), 5.21 (br s, 3 H) , 4.98 (br d, J=10.26 Hz, 3 H), 4.72 (br s, 1 H), 4.54 (br d, J=7.88 Hz, 3 H), 4.03 (br s, 9 H), 3.74 - 3.94 (m, 9 H), 3.45 - 3.71 (m, 12 H), 3.40 (br s, 6 H), 3.24 (br s, 7 H), 3.07 (br d, J=14.13 Hz, 5 H), 2.91 - 3.01 (m, 1 H), 2.24 - 2.44 (m, 5 H), 2.20 (br s, 1 H), 2.10 (s, 9 H), 1.96 - 2.04 (m, 9 H), 1.89 (br s , 9 H), 1.74 - 1.81 (m, 9 H), 1.51 - 1.73 (m, 6 H), 1.07 - 1.36 (m, 3 H). MS (ESI) [M+H] ⁺ m/z: 848.0 .

To a solution of compound VIII (200 g, 118 mmol, 1.00 eq. ) and tetrazole diisopropylammonium (8.08 g, 47.2 mmol, 0.40 eq. ) in dichloromethane (2.0 L) was added 3-bis(diisopropyl ammonium) (53.3 g, 177 mmol, 1.50 eq.), the reaction solution was stirred at 40°C for 2 hours, and LCMS showed that the conversion of compound 13 was completed. The reaction solution was washed with a 1:1 mixture of saturated NaHCO3 and saturated brine (2.0 L), dried over anhydrous Na ₂ SO ₄ , and the filtrate was concentrated. The crude product obtained was dissolved in dichloromethane (1.2 L), and was added dropwise to the stirring formazan. In tert-butyl ether (6.0 L), filter the suspension, rinse the filter cake with tert-butyl ether, collect the solid and dry it under vacuum, dissolve the product in dichloromethane (1.0 L) and concentrate to dryness, repeat Operate 4 times to remove residual tert-butyl ether to obtain GLPA15 (164 g, yield 73.3%). ¹ H NMR (400 MHz in DMSO-d6): δ ppm 8.05 (br d, J = 6.50 Hz, 2 H), 7.81 (br d, J=9.01 Hz, 3 H), 5.22 (d, J=3.25 Hz , 3 H), 4.98 (dd, J=11.26, 3.25 Hz, 3 H), 4.55 (br d, J=8.50 Hz, 3 H), 4.03 (s, 9 H), 3.64 - 3.97 (m, 12 H ), 3.55 - 3.63 (m, 6 H), 3.50 (br s, 5 H), 3.40 (br d, J=6.13 Hz, 6 H), 3.17 - 3.30 (m, 9 H), 3.07 (br d, J=14.26 Hz, 4 H), 2.76 (t, J=5.82 Hz, 2 H), 2.18 - 2.47 (m, 6 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 ( s, 9 H), 1.78 (s, 9 H), 1.52 - 1.74 (m, 6 H), 1.12 - 1.19 (m, 12 H). 31P NMR (DMSO-d6): ppm δ 145.25. MS (ESI) [M+H] ⁺ m/z: 1895.7.

In some studies, methods are provided for attaching a targeting group containing GalNAc (also referred to herein as a GalNAc delivery compound) to the 5'-end of the sense strand, which involves the last coupling in the solid phase synthesis The step uses GalNAc phosphoramidite (GLPA1), using a synthetic 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 5'-end of the sense strand.

In some studies, methods to attach a GalNAc-containing targeting group to the 3'-end of the sense strand include the use of a GLO-n-containing solid support (CPG). In some studies, methods to connect a GalNAc-containing targeting group to the 3'-end of the sense strand include: connecting the GalNAc targeting group to a CPG solid support via an ester bond and using it when synthesizing the sense strand. The resulting CPG has a GalNAc targeting group attached, 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 methods similar to this article or well-known in the art after using reasonably corresponding intermediates, and can be connected to the appropriate position of the siRNA duplex as a targeting group. Example 4. In vitro screening of C3 siRNA duplexes

Huh7 cells were trypsinized and adjusted to the appropriate density, then seeded into 96-well plates. Cells were transfected with test siRNA or PBS control using Lipofectamine RNAiMax (Invitrogen-13778-150) according to the manufacturer's recommendations at the same time as seeding. siRNA was tested in triplicate at two concentrations (0.08 nM and 0.6 nM).

Twenty-four hours after transfection, the medium was removed and cells were harvested for RNA extraction. Total RNA was extracted using TRIzol™ Reagent (Invitrogen - 15596018) according to the manual.

cDNA was synthesized using PrimeScript™ RT Reagent Kit and gDNA Eraser (Perfect Real Time) (TaKaRa-RR047A) according to the manual. Detection of C3 cDNA 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. data analysis

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).

The equations are listed below:

ΔCT = average Ct of target gene – average Ct of GAPDH

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%.

Table 4 provides experimental results from in vitro studies using various C3 RNAi reagents to inhibit C3 expression; the double-stranded sequences used correspond to the compounds shown in Table 2. Duplex AV# Average inhibition % SD 0.6nM 0.08nM 0.6nM 0.08nM AV00375 89.66 80.97 0.88 1.51 AV00376 80.04 54.22 2.07 2.23 AV00377 86.27 71.81 1.75 2.28 AV00378 80.15 63.16 2.56 0.47 AV00379 48.04 28.03 3.85 3.00 AV00380 70.72 34.00 0.50 2.21 AV00381 32.56 9.04 4.96 7.84 AV00382 78.79 47.83 1.57 8.15 AV00383 50.68 11.08 0.60 4.03 AV00384 89.05 69.34 0.71 0.47 AV00385 75.08 29.64 1.35 3.48 AV00386 80.74 42.84 0.91 6.38 AV00387 33.36 13.81 0.96 11.29 AV00388 82.28 43.11 1.73 2.82 AV00389 85.69 68.12 1.21 1.46 AV00390 37.86 19.06 6.76 2.12 AV00391 11.49 7.59 12.73 3.46 AV00392 2.02 3.70 10.32 7.62 AV00393 83.04 56.71 0.79 3.29 AV00394 85.63 64.42 0.79 1.83 AV00395 20.75 6.46 3.40 6.63 AV00396 69.63 34.06 2.17 10.10 AV00398 56.44 -3.43 1.99 16.16 AV00399 23.19 -8.57 3.15 5.70 AV00400 60.97 6.35 0.89 9.96 AV00401 77.72 34.42 0.57 6.28 AV00402 77.35 39.88 2.42 2.48 AV00403 82.03 52.63 1.97 4.79 AV00404 57.67 28.05 6.24 3.47 AV00405 83.20 63.73 1.10 2.22 AV00406 68.52 41.76 5.85 2.35 AV00407 63.63 34.38 4.98 6.25 AV00408 84.43 65.55 1.20 3.07 AV00409 80.66 51.10 0.99 2.82 AV00410 78.96 49.11 1.33 1.38 AV00411 80.37 48.33 1.77 3.79 AV00412 -1.19 -6.73 5.97 9.11 AV00413 89.53 78.21 1.28 2.87 AV00415 87.26 63.52 1.03 3.37 AV00416 88.71 70.41 0.80 1.91 AV00417 68.29 27.12 2.06 7.42 AV00418 80.00 38.08 2.05 5.82 AV00419 72.57 25.79 1.42 4.35 AV00420 70.86 34.66 0.77 4.97 AV00421 87.21 72.95 1.24 1.36 AV00422 79.68 54.44 0.76 1.92 AV00423 62.35 39.20 3.87 0.96 AV00424 75.91 44.48 1.07 3.04 AV00425 76.20 39.92 1.91 6.36 AV00426 -10.91 -2.67 16.19 11.34 AV00427 64.92 24.72 2.33 5.76 AV00428 70.43 30.55 0.91 7.23 AV00429 90.36 77.31 0.72 1.21 AV00430 64.39 17.35 1.50 7.16 AV00431 31.89 19.58 5.11 10.32 AV00432 48.55 12.39 1.42 5.02 AV00433 86.15 50.08 0.86 4.57 AV00434 21.52 3.27 12.34 11.77 AV00435 76.63 51.50 4.78 4.80 AV00436 37.03 21.81 15.57 4.56 AV00437 51.27 17.90 0.47 8.64 AV00438 -14.06 -3.49 21.52 4.61 AV00439 62.79 23.11 1.35 5.13 AV00440 57.88 14.89 4.42 4.49 AV00441 67.25 31.15 6.02 7.40 AV00442 1.29 -7.59 4.94 10.62 AV00443 80.05 44.67 0.52 1.70 AV00444 31.00 -1.99 5.77 8.53 AV00445 22.94 -6.56 4.02 4.12 AV00446 31.22 -5.35 3.83 10.14 AV00447 18.12 -5.32 4.16 11.05 Example 5. In vivo testing of C3 siRNA duplexes

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). Fourteen 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 injected subcutaneously with a single dose of 3 mg/kg or 6 mg/kg C3 siRNA reagent or PBS. Blood samples were collected before siRNA administration on day 0 and at the end of days 7 and 14. Measure luciferase activity. By comparing the luciferase activity of blood samples before administration in the siRNA-treated group and the luciferase activity of blood samples collected on days 7 and 14 and normalized based on the luciferase activity changes in serum samples from the PBS-treated group , to calculate the remaining percentage, and the results are shown in Table 5.

Table 5. GLX-n in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958−16961, using compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 3. Delivery compounds represented by GalNAc3. Duplex ID The remaining percentage of luciferase on day 7 The remaining percentage of luciferase on day 14 Dosemg/kg Mean±SD Mean±SD AD00343 0.39±0.15 0.32±0.05 6.0 AD00344 0.82±0.36 0.46±0.04 6.0 AD00388 0.67±0.27 0.43±0.11 6.0 AD00389 0.49±0.45 0.32±0.13 6.0 AD00416 0.82±0.43 0.59±0.18 6.0 Example 6. In vivo testing of C3 siRNA duplexes

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 injected subcutaneously with a single dose of 3 mg/kg C3 siRNA reagent or PBS. Blood samples were collected before siRNA administration on day 0 and at the end of day 7 by siRNA-treated group and PBS-treated group on day 7, and normalized coding in plasma samples was measured relative to pre-treatment changes using an ELISA assay Human C3 protein concentration to calculate retention percentage; the results are shown in Table 6-8.

Table 6. GLX-n in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958−16961, using compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 3. Delivery compounds represented by GalNAc3. Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD) Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD) AD00345 0.48±0.14 AD00349 0.98±0.27 AD00346 0.40±0.14 AD00350 0.87±0.15 AD00347 0.26±0.04 AD00351 0.51±0.20 AD00348 0.39±0.06

Table 7. GLX-n Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136 using compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 3, 3 mg/kg each. , delivery compound represented by GalNAc3 in 16958−16961. Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD) Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD) AD00385 0.9 ±0.41 AD00392 1.25 ±0.5 AD00386 1.09 ±0.59 AD00393 0.92 ±0.39 AD00387 2.16±0.98 AD00394 1.01±0.35 AD00388 0.65 ±0.29 AD00395 0.39±0.68 AD00389 0.38 ±0.13 AD00396 0.37 ±0.71 AD00390 0.9 ±0.41 AD00397 0.95 ±0.42 AD00391 0.98 ±0.46

Table 8. GLX-n Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, using compounds corresponding to the sequences, chemical modifications, and delivery shown in Table 3, 3 mg/kg each, GLX-n , delivery compound represented by GalNAc3 in 16958−16961. Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD) Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD) AD00410 0.93 ±0.33 AD00416 0.13 ±0.03 AD00411 0.98 ±0.52 AD00417 0.22 ±0.12 AD00412 0.34 ±0.09 AD00418 0.35 ±0.05 AD00413 0.43 ±0.15 AD00419 0.31 ±0.12 AD00414 0.53 ±0.21 AD00420 0.76 ±0.21 AD00415 0.57 ±0.13 Example 7. In vivo testing of C3 siRNA duplexes

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 injected subcutaneously with a single dose of 3 mg/kg or 6 mg/kg 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 from the siRNA-treated group and the PBS-treated group on days 7, 14, and 21, and ELISA assay was used to measure the relative Pre-process changes were performed to normalize the concentration of the encoded human C3 protein in the plasma sample to calculate retention percentage; the results are shown in Table 9-11.

Table 9. Percent retention of human C3 in mouse plasma measured by ELISA for a single use of 3mpk compounds corresponding to the sequence, chemical modification, and delivery shown in Table 3. Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD) Day 7 Day 14 Day 21 AD00385 0.59±0.23 0.48±0.08 1.01±0.38 AD00579 1.19±0.63 0.7±0.31 1.53±0.68 AD00580 0.51±0.26 0.59±0.43 0.92±0.37 AD00581 0.33±0.05 0.5±0.12 0.89±0.53 AD00582 1.15±0.34 0.78±0.37 0.74±0.39 AD00583 1.66±1.15 1.17±0.19 0.62±0.38 AD00393 1.23±0.56 1.15±0.71 0.82±0.36 AD00584 1.74±0.96 0.84±0.27 0.89±0.29 AD00394 1.9±0.77 1.79±1.36 0.85±0.4 AD00527 1.99±1 1.61±0.53 0.92±0.14 AD00530 2.34±1.21 1.36±0.96 1.44±0.69 AD00531 1.74±0.98 1.71±0.82 1.19±0.35 AD00534 1.53±0.41 1.58±0.93 1.33±0.53

Table 10. Percent retention of human C3 in mouse plasma measured by ELISA for a single use of 6 mpk compounds corresponding to the sequence, chemical modification, and delivery shown in Table 3. Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD Day 7 Day 14 AD00343 0.68 ± 0.34 0.86 ± 0.74 AD00344-1 0.37 ± 0.11 0.68 ± 0.14 AD00388-1 0.45 ± 0.27 0.74 ± 0.61 AD00389-1 0.29 ± 0.09 0.53 ± 0.2 AD00580 0.2±0.09 0.54 ± 0.45 AD00581 0.38 ± 0.18 0.52 ± 0.2 AD00416 0.45±0.3 0.15 ± 0.05 AD00348 0.46 ± 0.32 0.23 ± 0.07 AD00351 0.68 ± 0.48 0.32 ± 0.16 AD00350 0.54 ± 0.19 0.7±0.25 AD00345 0.69 ± 0.44 0.48 ± 0.22 AD00740 1.53 ± 0.57 1.02±0.74 AD00741 0.43 ± 0.06 0.24 ± 0.13 AD00742 0.57 ± 0.18 0.3 ± 0.14 AD00743 0.5 ± 0.32 0.27 ± 0.04 AD00744 0.48 ± 0.3 0.17 ± 0.06

Table 11. Percent retention of human C3 in mouse plasma measured by ELISA for a single use of 6 mpk compounds corresponding to the sequence, chemical modification, and delivery shown in Table 3. Duplex ID Percent retention of human C3 in mouse plasma measured by ELISA (mean ± SD Day 7 Day 14 AD00385-1 1.03±0.18 0.69 ± 0.30 AD00416-1 0.60 ± 0.22 0.64 ± 0.27 equivalent

Although several embodiments of the invention have been described and illustrated herein, those of ordinary skill in the art will readily appreciate that various other methods may be used to perform the functions described herein and/or to obtain the results and/or one or more advantages. Each of these changes and/or modifications, means and/or structure, and such changes and/or modifications are deemed to be within the scope of the 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 use of the teachings of the present invention. Application. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood, therefore, that the foregoing embodiments are presented by way of example only and within the scope of the appended claims and their equivalents, the 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. Furthermore, any combination of two or more such features, systems, articles, materials and/or methods is also included in the invention provided that such features, systems, articles, materials and/or methods are not inconsistent with each other. within the range.

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.

Where a quantitative limitation is not used in the description and patent application, it shall be understood as "at least one" unless explicitly stated to the contrary.

The phrase "and/or" as used in the specification and claims should be understood to mean "one or both" of the elements so combined that such elements appear in combination in some cases and separately in other cases. . In addition to elements specifically identified by "and/or", other elements may optionally be present, whether related or unrelated to those specifically identified elements, unless expressly stated to the contrary.

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

without

without

without.

Claims (69)

A double-stranded ribonucleic acid (dsRNA) reagent for inhibiting the expression of complement component C3, wherein the dsRNA reagent includes a sense strand and an antisense strand, and nucleotide positions 2 to 18 in the antisense strand include those of C3 RNA A region of transcripts that is complementary, wherein the complementary region contains at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, and any Targeting ligands are optionally included. The dsRNA reagent of claim 1, wherein the region complementary to the C3 RNA transcript includes at least 15, 16, 17, 18 or 19 consecutive nucleotides that are consistent with those listed in one of Tables 1-3 One of the antisense sequences differs by no more than 3 nucleotides. The dsRNA reagent of claim 1 or 2, wherein the antisense strand of the dsRNA is at least substantially complementary to any target region in SEQ ID NO: 1, and is provided in any one of Tables 1-3. The dsRNA reagent of claim 3, wherein the antisense strand of the dsRNA is completely complementary to any target region in SEQ ID NO: 1 and is provided in any one of Tables 1-3. The dsRNA reagent according to claim 1, wherein the dsRNA reagent includes the sense strand sequence described in any one of Tables 1-3, wherein the sense strand sequence is at least substantially the same as the antisense strand sequence in the dsRNA reagent. complement each other. The dsRNA reagent according to claim 1, wherein the dsRNA reagent includes the sense strand sequence described in any one of Tables 1-3, wherein the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA reagent. . The dsRNA reagent according to claim 1, wherein the dsRNA reagent contains the antisense strand sequence listed in any one of Tables 1-3. The dsRNA reagent of claim 1, wherein the dsRNA reagent includes a sequence listed as a duplex sequence in any one of Tables 1-3. The dsRNA reagent of claim 1, wherein the dsRNA reagent contains at least one modified nucleotide. The dsRNA reagent of claim 1, wherein all or substantially all nucleotides in the antisense strand are modified nucleotides. The dsRNA reagent of claim 9 or 10, wherein the at least one modified nucleotide includes: 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide , 2'3'-seco nucleotide mimics, locked nucleotides, open nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabinose nucleotides, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, reverse nucleotide, reverse abasic nucleotide, reverse 2'-OMe nucleotide, reverse 2 '-deoxynucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide and 3'-OMe nucleotide, containing 5'-phosphorothioate group group of nucleotides, or terminal nucleotides linked to cholesterol derivatives or dodecyl dodecylamide groups, 2'-amino modified nucleotides, phosphoramidates, or non-nucleotide-containing nucleotides. Natural base. The dsRNA reagent of claim 9 or 10, wherein E-vinyl phosphonate nucleotide is included at the 5' end of the guide strand. The dsRNA reagent of claim 1, wherein the dsRNA reagent contains at least one phosphorothioate internucleoside linkage. The dsRNA reagent of claim 1, wherein the sense strand comprises at least one phosphorothioate internucleoside linkage. The dsRNA reagent of claim 1, wherein the antisense strand comprises at least one phosphorothioate internucleoside linkage. The dsRNA reagent of claim 1, wherein the sense strand comprises 1, 2, 3, 4, 5 or 6 phosphorothioate internucleoside linkages. The dsRNA reagent of claim 1, wherein the antisense strand comprises 1, 2, 3, 4, 5 or 6 phosphorothioate internucleoside linkages. The dsRNA reagent of claim 1, wherein all or substantially all nucleotides of the sense strand and antisense strand are modified nucleotides. The dsRNA reagent of claim 1, wherein the modified sense strand is a modified sense strand sequence listed in one of Tables 2-3. The dsRNA reagent of claim 1, wherein the modified antisense strand is a modified antisense strand sequence listed in one of Tables 2-3. The dsRNA reagent of claim 1, wherein the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is 16 to 23 nucleotides. The dsRNA reagent of claim 21, wherein the length of the complementary region is 19 to 21 nucleotides. The dsRNA reagent as described in claim 1, wherein the length of each strand is no more than 30 nucleotides. The dsRNA reagent of claim 1, wherein the length of each strand is no more than 25 nucleotides. The dsRNA reagent of claim 1, wherein the length of each strand is no more than 23 nucleotides. The dsRNA reagent of claim 1, wherein the dsRNA reagent contains at least one modified nucleotide, and further contains one or more targeting groups or linking groups. The dsRNA reagent of claim 26, wherein the one or more targeting groups or linking groups are conjugated to the sense strand. The dsRNA reagent of claim 26 or 27, wherein the targeting group or linking group includes N-acetyl-galactosamine (GalNAc). The dsRNA reagent according to claim 28, wherein the targeting group or linking group has a structure represented by formula (X), including a targeting moiety, a linking bond and a linker W, wherein the targeting moiety is selected from N- Acetyl-galactosamine derivative (GalNAc), which is linked to the linker W through a linking bond, the linker W has a structure shown in formula (XI), X is selected from O, NH ₂ or S, Y ^- is selected from: O ^﹣ , S ^﹣ , methyl or NRaRb, Ra and Rb are independently selected from hydrogen, substituted or unsubstituted C ₁ -C ₆ alkyl group, substituted or unsubstituted C ₃ -C ₆ cycloalkyl group, Or Ra and Rb are linked together with the attached atoms to form a 3-12 membered heterocycloalkyl group containing 1-3 N, O, S heteroatoms; preferred substituted or unsubstituted C ₁ -C ₆ alkyl groups, In the substituted or unsubstituted C ₃ -C ₆ cycloalkyl group, the substituent is selected from hydroxyl and amino; Formula (X) (Formula XI). The dsRNA reagent according to claim 29, wherein the linking bond in the targeting group is selected from polyethylene glycol, optionally substituted C ₂ -C ₁₂ alkyl, substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl group, substituted or unsubstituted C ₃ -C ₁₂ heterocycloalkyl group, substituted or unsubstituted C ₃ -C ₁₂ amide. The dsRNA reagent of claim 30, wherein substituted or unsubstituted C ₂ -C ₁₂ alkyl, substituted or unsubstituted C ₃ -C ₁₂ cycloalkyl, substituted or unsubstituted C ₃ -C ₁₂ heterocycle In alkyl, substituted or unsubstituted C ₃ -C ₁₂ amide, the substituent is selected from hydroxyl and carbonyl. The dsRNA reagent according to claim 30, wherein the linking bond in the targeting group is preferably from the following fragments: , , , , , and , where each m is independently an integer from 1 to 6, each n, o, p is independently 0 or 1, and each q ¹ and q ² are independently 0, 1 or 2. The dsRNA reagent as claimed in claim 32, wherein the linking bond in the targeting group is more preferably from the following fragments: , , , , , . The dsRNA reagent as described in claim 29, wherein the targeting part in the targeting group has the following structural fragment, n' is 1 or 2. The dsRNA reagent as described in claim 29, wherein X is selected from O or S, and Y ^- is selected from: O ^- or S ^- . The dsRNA reagent according to claim 26 or 27, wherein the targeting group has the following structure: GLO-1 GLS-1 GLO-2 GLS-2 GLO-3 GLS-3 GLO-4 GLS-4 GLO-5 GLS-5 GLO-6 GLS-6 GLO-7 GLS-7 GLO-8 GLS-8 GLO-9 GLS-9 GLO-10 GLS-10 GLO-11 GLS-11 GLO-12 GLS-12 GLO-13 GLS-13 GLO-14 GLS-14 GLO-15 GLS-15 GLO-16 GLS-16. The dsRNA reagent of claim 1, wherein the dsRNA reagent comprises a targeting group conjugated to the 5'-end of the sense strand. The dsRNA reagent of claim 1, wherein the dsRNA reagent comprises a targeting group conjugated to the 3'-end of the sense strand. The dsRNA reagent of claim 1, wherein the antisense strand contains a reverse abasic residue at the 3'-end. The dsRNA reagent of claim 1, wherein the sense strand contains one or two reverse abasic residues at the 3' or/and 5' end. The dsRNA reagent according to claim 1, wherein the dsRNA reagent has two blunt ends. The dsRNA reagent of claim 1, wherein at least one strand includes a 3' overhang of at least 1 nucleotide. The dsRNA reagent of claim 1, wherein at least one strand includes a 3' overhang of at least 2 nucleotides. The dsRNA reagent as described in claim 1, the sense strand has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3'-end and/or 5'-end. protruding end. A double-stranded ribonucleic acid (dsRNA) reagent for inhibiting the expression of complement component C3, wherein the dsRNA reagent contains a sense strand and an antisense strand, wherein the length of each strand is 14 to 30 nucleotides, and the antisense strand is The sense strand contains a region complementary to the C3 RNA transcript at nucleotide positions 2 to 18, wherein the complementary region contains 0, 1, 2, or 3 nucleotides that differ from one of the antisense sequences listed in Formula I At least 15 contiguous nucleotides of the acid, and optionally including a targeting ligand, double-stranded ribonucleic acid (dsRNA) reagent antisense strand: 5'-N _a -(A ₁ A ₂ A ₃ A ₄ )iN _b –(B ₁ B ₂ B ₃ B ₄ ) -N _b -(C ₁ C ₂ C ₃ C ₄ C ₅ )j -N _a - 3' where: i and j are independently selected from 0 or 1; each N _a and N _b independently represent 0-17 oligonucleotides; A ₁ A ₂ A ₃ A ₄ represents four consecutive nucleotides, which are represented in turn by lowercase 2'-OMe, uppercase 2'-Fluoro, and lowercase 2' -OMe, a motif modified by lowercase 2'-OMe, further including phosphorothioate internucleoside linkages between A ₁ and A ₂ and between A ₂ and A ₃ ; B ₁ B ₂ B ₃ B ₄ Represents a motif in which four consecutive nucleotides are all modified with lowercase 2'-OMe; C ₁ C ₂ C ₃ C ₄ C ₅ represents a motif in which five consecutive nucleotides are all modified with lowercase 2'-OMe The sequence further includes a phosphorothioate internucleoside linkage between _C3 and _C4 and between _C4 and _C5 . For the dsRNA reagent described in claim 45, N _a independently represents 0 oligonucleotides, N _b independently represents 2-5 chemically modified oligonucleotides, and i and j represent 1 respectively. The dsRNA reagent of claim 45, wherein the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 1. For the dsRNA reagent described in claim 45, the antisense strand of the dsRNA is completely complementary to any target region of SEQ ID NO: 1. . The dsRNA reagent of claim 45, wherein the sense strand sequence of any one of the dsRNA is at least substantially complementary to the antisense strand sequence in the dsRNA reagent. The dsRNA reagent according to claim 45, wherein the sense strand sequence of any one of the dsRNA is completely complementary to the antisense strand sequence in the dsRNA reagent. The dsRNA reagent of claim 45, wherein the dsRNA sense strand contains one or two reverse abasic residues at the 3' or/and 5' end. The dsRNA reagent of claim 45, wherein the dsRNA has two blunt ends. The dsRNA reagent of claim 45, wherein the dsRNA sense strand has a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 at the 3'-end and/or 5'-end nucleotide overhang. A composition comprising the dsRNA agent of any one of claims 1 to 53. The composition of claim 54, further comprising a pharmaceutically acceptable carrier. The composition of claim 55, further comprising one or more additional therapeutic agents. The composition of claim 56, wherein the composition is packaged in a kit, container, wrapper, dispenser, prefilled syringe or vial. The composition of claim 54, wherein the composition is formulated for subcutaneous administration or is formulated for intravenous (IV) administration. A cell comprising the dsRNA agent of any one of claims 1 to 53. The cell of claim 59, wherein the cell is a mammalian cell, optionally a human cell. A method for inhibiting the expression of complement component C3 gene in cells, which includes: (i) Preparing cells comprising an effective amount of the double-stranded ribonucleic acid (dsRNA) agent as described in any one of claims 1 to 53 or the composition as described in any one of claims 54 to 58; (ii) Maintain the cells prepared in (i) for a sufficient time to obtain degradation of the mRNA transcript of the C3 gene, thereby inhibiting the expression of the C3 gene in the cells. The method of claim 61, wherein the cells are in a subject and the dsRNA agent is administered subcutaneously to the subject. A method of inhibiting C3 gene expression in a subject, wherein the method comprises administering to the subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent as described in any one of claims 1 to 53 or as claimed in claims 54 to 58 The composition of any one. A method of treating a disease or disorder associated with C3 protein, wherein the method comprises administering to a subject an effective amount of a double-stranded ribonucleic acid (dsRNA) agent as described in any one of claims 1 to 53 or as claimed in claim 54 The composition of any one of to 58 to inhibit C3 gene expression. The method of claim 64, wherein the disease or disorder is caused by or related to: C3 activation or symptoms or progression thereof in response to C3 activation, the disease or disorder is typically associated with C3 expression, the disease Includes one or more of the following: 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), severe Myasthenia (MG), primary membranous nephropathy, macular degeneration (AMD), rheumatoid arthritis (RA), antineutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), periodontal disease and periodontal disease, malarial anemia, sepsis, and neurodegenerative diseases. The method of claim 65, further comprising administering an additional treatment regimen to the subject. The method of claim 66, wherein the additional treatment regimen includes administering to the subject one or more C3 antisense polynucleotides and administering to the subject a non-C3 dsRNA therapeutic. The method of claim 67, wherein the non-C3 dsRNA therapeutic agent is one or more of the following: additional therapeutic agents, such as anti-complement component C5 antibodies or antigen-binding fragments thereof, iRNA targeting complement components C5 and/or C3 peptide inhibitors. The method of claim 68, wherein the non-C3 dsRNA therapeutic agent is selected from one or more of the following: eculizumab or comprosimvastatins.