Complement component c3 irna composition and use methods thereof

Abstract

To provide alternative therapies and combination therapies for subjects having a complement component C3 associated disease.SOLUTION: The invention relates to RNA agents targeting complement component C3 gene (C3), for example, double stranded RNA (dsRNA) agents. The invention also relates to methods of using such RNAi agents for inhibiting expression of a C3 gene, and for preventing and treating C3 associated disorders, e.g., cold agglutinin disease (CAD), warm autoimmune hemolytic anemia, and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), bullous pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 62 / 924,210, filed October 22, 2019, the contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on October 16, 2020, is named 121301_10520_SL.txt, and is 1,327,438 bytes in size. [Background technology]

[0003] Background of the Invention Complement was first discovered in the 1890s and was found to assist, or "complement," the bactericidal activity of heat-stable antibodies present in normal serum at that time (Walport, MJ (2001) N Engl J Med. 344:1058). The complement system consists of more than 30 proteins present in the blood as soluble or membrane-associated proteins. Complement activation leads to a sequential cascade of enzymatic reactions, known as the complement activation pathway, that result in the formation of the potent anaphylatoxins C3a and C5a, which induce a wide range of physiological responses, ranging from chemoattraction to apoptosis. Initially, complement was thought to provide a robust and rapid response against invading pathogens and play a key role in innate immunity. However, in recent years, increasing evidence has suggested that complement is involved in adaptive immunity, involving T and B cells in pathogen elimination (Dunkelberger JR and Song WC. (2010) Cell Res. 20:34; Molina H, et al. (1996) Proc Natl Acad Sci U S A. 93:3357), plays an important role in immunological memory to prevent pathogen reinvasion, and is involved in many human disease states (Qu, H, et al. (2009) Mol Immunol. 47:185; Wagner, E. and Frank MM. (2010) Nat Rev Drug Discov. 9:43).

[0004] Complement is known to be activated through three distinct pathways: alternative, classical, and lectin (Figure 1), which involve proteins that exist largely as active zymogens and are then sequentially cleaved and activated.

[0005] The classical pathway is often activated by antibody-antigen complexes or C-reactive protein (CRP), both of which interact with the complement component C1q. Additionally, the classical pathway can be activated by phosphatidylserine present in apoptotic bodies in the absence of immune complexes.

[0006] The lectin pathway is initiated by mannose-binding lectin (MBL), which binds to complex carbohydrate residues on the surface of pathogens. Activation of the classical or lectin pathway leads to activation of the (C4b2b)C3 convertase.

[0007] The alternative pathway is activated by the binding of C3b, which is spontaneously produced by hydrolysis of C3 on targeted surfaces. This surface C3b is then recognized by factor B to form the complex C3bB. The C3bB complex is subsequently cleaved by factor D to form the active form of the AP, C3 convertase (C3bBb). Both types of C3 convertase cleave C3 to form C3b. C3b then further binds to factor B, either enhancing complement activation via the AP (the so-called alternative or amplification loop) or leading to the formation of active C5 convertase (C3bBbC3b or C4bC2bC3b), which cleaves C5 and triggers later events resulting in the formation of the membrane attack complex (MAC) (C5b-9).

[0008] Inappropriate activation of the complement system is responsible for propagating and / or initiating pathology in many different diseases, including, for example, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), myasthenia gravis (MG), C3 glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, ischemia-reperfusion injury, and neurodegenerative diseases. Summary of the Invention [Problem to be solved by the invention]

[0009] Therapies for the treatment of complement component C3-related diseases are limited and require time-consuming and expensive invasive procedures. Thus, there is a need in the art for alternative and combination therapies for subjects with complement component C3-related diseases. [Means for solving the problem]

[0010] Summary of the Invention The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of a gene encoding complement component C3. Complement component C3 can be in a cell, e.g., a cell within a subject, such as a human subject.

[0011] In certain embodiments, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of complement component C3 in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 0, 1, 2, or 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO:5 by no more than 1, 2, or 3 nucleotides.

[0012] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of complement component C3 in a cell, wherein the dsRNA comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region complementary to an mRNA encoding complement component C3, and the region of complementarity comprises at least 15 contiguous nucleotides that differ by 0, 1, 2, or no more than 3 nucleotides from any of the antisense nucleotide sequences in any of Tables 2-7, 15, 18, 20-23, 30, and 31.

[0013] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of complement component C3 in a cell, wherein the dsRNA comprises a sense strand and an antisense strand that form a double-stranded region, and wherein the sense strand is selected from nucleotides 475-497, 487-509, 490-512, 491-513, 705-727, 809-831, 813-835, 1147-1169, 1437-1459, 1439-1461, 1447-1469, 2596-2618, 2634-2656, 3012-3034, 3334-3356, 3611-3633, 3614-3635, 3615-3636, 3616-3637, 3617-3638, 3618-3639, 3620-3621, 3621-3622, 3622-3623, 3623-3624, 3624-3625, 3625-3626, 3626-3627, 3627-3628, 3628-3629, 3630-3631, 3632-3633, 3634-3635, 3636-3638, 3639-3640, 3641-3642, 3642-3643, 3643-3644, 3645-3646, 3646-3648, 3647-3649 the antisense strand comprises at least 15 contiguous nucleotides that differ by 0, 1, 2, or no more than 3 nucleotides from any of the nucleotide sequences of SEQ ID NO:6, 3622-3655, 3809-3831, 3846-3868, 3847-3869, 3920-3942, 4047-4069, 4061-4083, 4156-4178, 4157-4177, 4162-4184, 4178-4200, 4226-4248, 4369-4391, 4392-4414, 4521-4543, 4522-4544, 4523-4545, and 5012-5034, and the antisense strand comprises at least 19 contiguous nucleotides of the corresponding nucleotide sequence of SEQ ID NO:5.

[0014] In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence of any of nucleotides 705-727, 809-831, or 634-2656 of SEQ ID NO: 1. In other embodiments, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence of nucleotides 634-2656 of SEQ ID NO: 1.

[0015] In some embodiments, the antisense strand is AD-565541.2, AD-564742, AD-567304, AD-568978, AD-569164, AD-569272.2, AD-569765.2, AD-564730.2, AD-567315, AD-564745.2, AD-571715.2 , AD-570714, AD-571826, AD-572041.2, AD-572039.2, AD-572387, AD-568586.2, AD-5 66837.2, AD-566444.2, AD-567700.2, AD-567814.2, AD-568003.2, AD-569164.2, AD- 569763.2, AD-565281.2, AD-571539.2, AD-572389.2, AD-567315.2, AD-571752.2, AD-568026.2, AD-571298, AD-572110.2, AD-572062.2, AD-572388.2, AD-572040.2, AD-567713.2, AD-567521.2, AD-567066.2, AD-1181519, AD-569268, or AD-570714,

[0016] In certain embodiments, the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 0, 1, 2 or 3 nucleotides from any of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1181519, AD-569268, or AD-570714. In other embodiments, the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 0, 1, 2 or 3 nucleotides from the antisense strand nucleotide sequence of AD-570714.

[0017] In certain embodiments, the dsRNA agent includes at least one modified nucleotide.

[0018] In some embodiments, substantially all nucleotides of the sense strand; substantially all nucleotides of the antisense strand comprise the modification; or substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand comprise the modification.

[0019] In some embodiments, every nucleotide in the sense strand comprises a modification; every nucleotide in the antisense strand comprises a modification; or every nucleotide in the sense strand and every nucleotide in the antisense strand comprise a modification.

[0020] In certain embodiments, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'- O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural base-containing nucleotides, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, phosphorothioate-group-containing nucleotides, methylphosphonate-group-containing nucleotides, 5'-phosphate-containing nucleotides, 5'-phosphate-containing nucleotide mimics, thermally destabilized nucleotides, glycol-modified nucleotides (GNAs), and 2-O-(N-methylacetamide)-modified nucleotides; and combinations thereof.

[0021] In some embodiments, the modification of the nucleotide is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol; and combinations thereof.

[0022] In certain embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNAs), such as Ggn, Cgn, Tgn, or Agn, and vinyl-phosphonate nucleotides; and combinations thereof.

[0023] In other embodiments, at least one of the nucleotide modifications is a thermally destabilizing nucleotide modification.

[0024] In some embodiments, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification; a mismatch with the opposite nucleotide in a duplex; and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0025] The double-stranded region can be 19 to 30 nucleotide pairs in length; 19 to 25 nucleotide pairs in length; 19 to 23 nucleotide pairs in length; 23 to 27 nucleotide pairs in length; or 21 to 23 nucleotide pairs in length.

[0026] In some embodiments, each strand independently does not exceed 30 nucleotides in length.

[0027] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0028] The region of complementarity can be at least 17 nucleotides in length; 19 to 23 nucleotides in length; or 19 nucleotides in length.

[0029] In certain embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide, hi other embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0030] In some embodiments, the dsRNA agent further comprises a ligand.

[0031] In certain embodiments, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

[0032] In certain embodiments, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

[0033] In certain embodiments, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

[0034] In some embodiments, the ligand is [ka] is.

[0035] In certain embodiments, the dsRNA agent is conjugated to a ligand shown in the diagram below. [ka] wherein X is O or S.

[0036] In certain embodiments, X is O.

[0037] In certain embodiments, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0038] In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand, eg, the antisense strand or the sense strand.

[0039] In other embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand, eg, the antisense strand or the sense strand.

[0040] In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand. In certain embodiments, the strand is the antisense strand.

[0041] In certain embodiments, the first base pair at the 5' end of the antisense strand of the duplex is an AU base pair.

[0042] The invention also provides cells comprising any of the dsRNA agents of the invention and pharmaceutical compositions comprising any of the dsRNA agents of the invention.

[0043] A pharmaceutical composition of the invention can contain a dsRNA agent in an unbuffered solution, e.g., saline or water, or a pharmaceutical composition of the invention can contain a dsRNA agent in a buffer, e.g., a buffer containing acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof; or phosphate buffered saline (PBS).

[0044] In one embodiment, the present invention provides a method for inhibiting expression of the complement component C3 gene in a cell, comprising contacting the cell with any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby inhibiting expression of the complement component C3 gene in the cell.

[0045] In certain embodiments, the cell is in a subject, e.g., a human subject, e.g., a subject having a complement component C3-related disorder, such as a complement component C3-related disorder selected from the group consisting of cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

[0046] In certain embodiments, contacting a cell with a dsRNA agent inhibits expression of complement component C3 by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0047] In certain embodiments, inhibiting expression of complement component C3 reduces complement component C3 protein levels in the subject's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0048] In one embodiment, the present invention provides a method for treating a subject having a disorder that would benefit from reduced expression of complement component C3. The method comprises administering to the subject a therapeutically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby treating the subject having a disorder that would benefit from reduced expression of complement component C3.

[0049] In another aspect, the present invention provides a method for preventing at least one symptom in a subject having a disorder that would benefit from reduced expression of complement component C3. The method comprises administering to the subject a prophylactically effective amount of any of the dsRNAs of the present invention or any of the pharmaceutical compositions of the present invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduced expression of complement component C3.

[0050] In certain embodiments, the disorder is a complement component C3-associated disorder, e.g., the complement component C3-associated disorder is selected from the group consisting of cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

[0051] In certain embodiments, the complement component C3-associated disorder is cold agglutinin disease (CAD).

[0052] In certain embodiments, the subject is a human.

[0053] In certain embodiments, administration of the agent to a subject causes a decrease in hemolysis and / or a decrease in C3 protein accumulation.

[0054] In certain embodiments, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0055] In certain embodiments, the dsRNA agent is administered to the subject subcutaneously.

[0056] In certain embodiments, the methods of the invention further comprise determining the level of complement component C3 in a sample from the subject.

[0057] In certain embodiments, the level of complement component C3 in a subject sample is the level of complement component C3 protein in a blood or serum sample.

[0058] In certain embodiments, the methods of the present invention further comprise administering to the subject an additional therapeutic agent for the treatment of hemolysis.

[0059] The invention also provides kits comprising any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention and, optionally, instructions for use. [Brief explanation of the drawings]

[0060] [Figure 1] Schematic representation of the alternative, classical and lectin complement pathways.

[0061] [Figure 2] 1 is a graph showing C3 mRNA levels in mice (n=3 / group) 14 days after a single subcutaneous dose of 2 mg / kg of the indicated dsRNA duplex. C3 mRNA levels are shown relative to control levels detected with PBS treatment.

[0062] [Figure 3] 1 is a graph showing C3 mRNA levels in mice (n=3 / group) 14 days after a single subcutaneous dose of 2 mg / kg of the indicated dsRNA duplex. C3 mRNA levels are shown relative to control levels detected with PBS treatment.

[0063] [Figure 4] 1 is a graph showing C3 mRNA levels in mice (n=3 / group) 14 days after a single subcutaneous dose of 2 mg / kg of the indicated dsRNA duplex. C3 mRNA levels are shown relative to control levels detected with PBS treatment.

[0064] [Figure 5] 1 is a table showing treatment groups of cynomolgus monkeys receiving a single subcutaneous dose of 3 mg / kg or 25 mg / kg of the indicated dsRNA duplexes.

[0065] [Figure 6] Graph showing the effect of a single 3 mg / kg or 25 mg / kg subcutaneous dose of the indicated dsRNA duplex on residual % C3 protein levels normalized to the mean pre-dose C3 protein level in cynomolgus monkey serum. Baseline was adjusted to day 1 dosing for all groups.

[0066] [Figure 7] 1 is a table showing treatment groups of cynomolgus monkeys given a single 3 mg / kg subcutaneous dose of the indicated dsRNA duplex.

[0067] [Figure 8] 1 is a graph showing the effect of a single 3 mg / kg or 25 mg / kg subcutaneous dose of the indicated dsRNA duplex on residual % C3 protein levels normalized to the mean pre-dose C3 protein level in cynomolgus monkey serum.

[0068] [Figure 9] 1 is a table showing treatment groups and timing of dosing and biopsies for cynomolgus monkeys receiving a single dose of 3 mg / kg, 9 mg / kg, or 25 mg / kg or multiple doses of 3 mg / kg (3x3) of the indicated dsRNA duplexes.

[0069] [Figure 10]1 shows the effect of a single 3 mg / kg or 25 mg / kg subcutaneous dose of the indicated dsRNA duplex on the residual % C3 protein level normalized to the mean pre-dose C3 protein level in cynomolgus monkey serum. For Group 2, day -6 on the graph corresponds to day -27, and day 1 is the day the duplex was administered. DETAILED DESCRIPTION OF THE INVENTION

[0070] Detailed Description of the Invention The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the complement component C3 gene. The gene can be in a cell, for example, a cell in a subject, such as a human. The use of these iRNAs allows for targeted degradation of the mRNA of the corresponding gene (complement component C3 gene) in a mammal.

[0071] The iRNAs of the present invention are designed to target the human complement component C3 gene, including portions of the gene that are conserved with complement component C3 orthologs in other mammalian species. Without intending to be bound by theory, it is believed that combinations or subcombinations of the above properties and specific target sites or specific modifications in these iRNAs improve the efficacy, stability, potency, durability, and safety of the iRNAs of the invention.

[0072] Thus, the present invention provides methods for treating and preventing complement component C3-associated disorders, such as cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathies, using iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the complement component C3 gene.

[0073] The iRNA of the present invention comprises an RNA strand (antisense strand) having a region that is up to about 30 nucleotides in length or less, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least a portion of an mRNA transcript of the complement component C3 gene.

[0074] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, and have a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the complement component C3 gene. In certain embodiments, such RNA agents having a long antisense strand may include a second RNA strand (sense strand) preferably 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0075] The use of iRNAs of the present invention allows for targeted degradation of the mRNA of the corresponding gene (complement component C3 gene) in mammals. Using in vitro and in vivo assays, the inventors have shown that iRNAs targeting the C3 gene can potently mediate RNAi, resulting in significant inhibition of C3 gene expression. Therefore, methods and compositions containing these iRNAs are useful for treating subjects with complement component C3-related disorders, such as cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, including pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

[0076] Thus, the present invention provides methods and combination therapies for the treatment of subjects with disorders that would benefit from inhibition or reduction of C3 gene expression, e.g., complement component C3-associated diseases such as cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathies, using iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the C3 gene.

[0077] The present invention also provides methods for preventing at least one symptom in a subject with a disorder that would benefit from inhibiting or reducing C3 gene expression, e.g., cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

[0078] For example, in a subject with cold agglutinin disease (CAD), the method of the present invention can prevent at least one symptom in the subject, including, for example, hemolysis, MAC deposition and tissue damage, inflammation (e.g., chronic inflammation); in a subject with warm autoimmune hemolytic anemia, the method of the present invention can prevent at least one symptom in the subject, including, for example, hemolysis, inflammation (e.g., chronic inflammation), and MAC tissue damage; in a subject with paroxysmal nocturnal hemoglobinuria (PNH), the method of the present invention can prevent at least one symptom in the subject, including, for example, hemolysis, inflammation (e.g., chronic inflammation), thrombosis, and hematopoietic failure; in a subject with lupus nephritis (LN), the method of the present invention can prevent at least one symptom in the subject, including, for example, inflammation (e.g., chronic inflammation), hematuria In a subject with pemphigoid, the methods of the present invention may prevent at least one symptom in the subject, including, for example, blister formation, inflammation (e.g., chronic inflammation), C3 deposition, and MAC tissue damage; in a subject with pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), the methods of the present invention may prevent at least one symptom in the subject, including, for example, blister formation, inflammation (e.g., chronic inflammation), C3 deposition, and MAC tissue damage; and in a subject with C3 glomerulopathy, the methods of the present invention may prevent at least one symptom in the subject, including, for example, inflammation (e.g., chronic inflammation), hematuria, proteinuria, edema, hypertension, and renal failure.

[0079] The following detailed description discloses methods for making and using compositions comprising iRNA that inhibit expression of the complement component C3 gene, as well as compositions, uses, and methods for treating subjects who would benefit from inhibiting and / or reducing expression of the complement component C3 gene, e.g., subjects susceptible to or diagnosed as having a complement component C3-associated disorder.

[0080] I. Definition In order that the present invention may be better understood, certain terms are first defined. Furthermore, it should be noted that whenever a value or range of values ​​for a parameter is given, it is intended that values ​​and ranges intermediate to the given values ​​are also part of the present invention.

[0081] As used herein, the articles "a," "an," and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, for example, a plurality of elements.

[0082] The term "including" is used herein to mean, and is used interchangeably with, the term "including but not limited to."

[0083] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. For example, "the sense strand or the antisense strand" is understood as "the sense strand or the antisense strand, or the sense strand and the antisense strand."

[0084] The term "about" is used herein to mean within a typical acceptable range in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In some embodiments, about means ±10%. In some embodiments, about means ±5%. When about precedes a series of numerical values ​​or ranges, it is understood that "about" can modify each numerical value in the series or range.

[0085] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that are logically included from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides of a 21-nucleotide nucleic acid molecule" means that 19, 20, or 21 nucleotides have the property indicated. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each number in the series or range.

[0086] As used herein, "less than" or "less than" refers to the value adjacent to the term and the logically lower value or integer up to 0, depending on the context. For example, a duplex having an overhang of "two or fewer nucleotides" has 2, 1, or 0 nucleotide overhangs. When "less than" precedes a series of numbers or ranges, it is understood that "less than" can modify each number in the series or range. As used herein, ranges include both upper and lower limits.

[0087] In the event of a discrepancy between the sequence and a designated position in a transcript or other sequence, the nucleotide sequence set forth herein will control.

[0088] As used herein, the term "complement component 3" is used interchangeably with the term "C3" and refers to a well-known gene and polypeptide that is also known in the art as ARMD9, C3a anaphylatoxin, ASP, complement component C3a, C3a, complement component C3b, C3b, prepro-C3, acylation-stimulated protein cleavage product, CPAMD1, complement C3, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1, complement component C3, and AHUS5. The term "C3" includes human C3, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. NM_000064.3 (GI:726965399; SEQ ID NO:1); mouse C3, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. NM_009778.3 (GI:773669943; SEQ ID NO:2); and rat C3, whose amino acid and nucleotide sequences can be found, for example, in GenBank Accession No. NM_016994.2 (GI:158138560; SEQ ID NO:3).

[0089] The term "C3" also includes cynomolgus C3, the amino acid and nucleotide sequences of which can be found, for example, in GenBank Accession No. XM_005587719.2 (GI:982312947; SEQ ID NO:4) and in gene entry ENSP00000245907 (Locus=chr19:6921416:6963034) at the Macaca genome project website (http: / / macaque.genomics.org.cn / page / species / index.jsp).

[0090] Further examples of C3 mRNA sequences are readily available using, for example, GenBank, UniProt, OMIM and the Macaca genome project web site.

[0091] Examples of C3 nucleotide sequences can be found in SEQ ID NOs: 1 to 8. SEQ ID NOs: 5 to 8 are the reverse complements of SEQ ID NOs: 1 to 4, respectively.

[0092] Further information about C3 is provided in NCBI Gene at, for example, http: / / www.ncbi.nlm.nih.gov / gene / 718.

[0093] The GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0094] As used herein, the terms "complement component C3" and "C3" also refer to naturally occurring DNA sequence variants of the C3 gene. Numerous sequence variants within the C3 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, e.g., http: / / www.ncbi.nlm.nih.gov / snp?LinkName=gene_snp&from_uid=718, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0095] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the complement component C3 gene, including mRNA, which is the RNA processing product of the primary transcription product.The target portion of the sequence is at least long enough to serve as a substrate for iRNA-specific cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the complement component C3 gene.In some embodiments, the target sequence is within the protein coding region of complement component C3.

[0096] The target sequence can be about 19-36 nucleotides in length, for example, preferably about 19-30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths between the above ranges and lengths are also considered part of the present invention.

[0097] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence using standard nucleotide nomenclature.

[0098] "G", "C", "A", "T" and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases, respectively. However, it is understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or alternative replacement moieties, which will be further detailed below (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially changing the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, nucleotides containing inosine as bases can base pair with nucleotides containing adenine, cytosine or uracil. Therefore, nucleotides containing uracil, guanine or adenine can be substituted with nucleotides containing inosine, for example, in the nucleotide sequence of dsRNA of the present invention. In another example, adenine and cytosine in any part of an oligonucleotide can be replaced with guanine and uracil, respectively, to form GU wobble base pairs with target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods of the present invention.

[0099] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent," as used interchangeably herein, include RNA, as that term is defined herein, and refer to agents that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, expression of the complement component C3 gene in cells, e.g., cells within a subject, such as a mammalian subject.

[0100] In some embodiments, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a complement component C3 target mRNA sequence, and directs the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into 19-23 base pair small interfering RNAs with characteristic 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding of the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one embodiment, the present invention relates to single-stranded RNA (siRNA) that is produced intracellularly and promotes RISC complex formation to silence a target gene, i.e., the complement component C3 gene. Thus, the term "siRNA" is also used herein to refer to the iRNA described above.

[0101] In some embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides long and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs described herein or chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

[0102] In some embodiments, the "iRNA" for use in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNA agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a ribonucleic acid molecule complex having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having a "sense" and an "antisense" orientation relative to the target RNA, i.e., the complement component C3 gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, for example, mRNA, through a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0103] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Furthermore, as used herein, "iRNA" may contain chemically modified ribonucleotides; iRNA may contain significant modifications to multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleotide includes, for example, the substitution, addition, or removal of a functional group or atom in the internucleoside linkage, sugar moiety, or nucleobase. Modifications suitable for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are included in the term "iRNA" or "RNAi agent" for purposes of this specification and claims.

[0104] The duplex region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can be about 19-36 base pairs in length, e.g., about 19-30 base pairs in length, e.g., about 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, or 36 base pairs in length, e.g., about 19-30, 19-29 , 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths between the above ranges and lengths are also considered to be part of the invention.

[0105] The two strands forming the duplex structure may be different portions of a single larger RNA molecule or may be separate RNA molecules. If the two strands are part of a single larger molecule, they are connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each other strand forming the duplex structure, and the connected RNA strands are referred to as "hairpin loops." A hairpin loop may contain at least one unpaired nucleotide. In certain embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In certain embodiments, a hairpin loop may be 10 or fewer nucleotides. In certain embodiments, a hairpin loop may be 8 or fewer unpaired nucleotides. In certain embodiments, a hairpin loop may be 4 to 10 unpaired nucleotides. In certain embodiments, a hairpin loop may be 4 to 8 nucleotides.

[0106] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, but do not necessarily, be covalently linked. When the two strands are covalently linked by any means other than an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each other strand forming a duplex structure, the connecting structure is called a "linker." The number of nucleotides in the RNA strands can be the same or different. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi can include one or more nucleotide overhangs.

[0107] In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which contains 19-23 nucleotides, that interacts with a target RNA sequence, eg, the complement component C3 gene, to direct cleavage of the target RNA.

[0108] In certain embodiments, the iRNA of the invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, eg, a complement component C3 target mRNA sequence, to direct cleavage of the target RNA.

[0109] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide extending from the duplex structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA can contain an overhang of at least one nucleotide; alternatively, the overhang can contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. The overhang can be in the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide can be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of a dsRNA.

[0110] In some embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' or 5' end. In some embodiments, the overhang of the sense strand or the antisense strand, or both strands, can be longer than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides in length. In some embodiments, the extended overhang is located on the sense strand of the duplex. In some embodiments, the extended overhang is located on the 3' end of the sense strand of the duplex. In some embodiments, the extended overhang is located on the 5' end of the sense strand of the duplex. In some embodiments, the extended overhang is located on the antisense strand of the duplex. In some embodiments, the extended overhang is located on the 3' end of the antisense strand of the duplex. In some embodiments, the extended overhang is located on the 5' end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the extended overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang contains a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0111] " Blunt " or " blunt end " means that there are no unpaired nucleotides at the end of the double-stranded RNA agent, i.e., there are no nucleotide overhangs. A "blunt-ended" double-stranded RNA agent is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The RNAi agent of the present invention includes RNAi agents that have no nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or no nucleotide overhangs at either end. In most cases, such molecules are double-stranded throughout their entire length.

[0112] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., complement component C3 mRNA.

[0113] As used herein, the term "region of complementarity" refers to a region of the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a complement component C3 nucleotide sequence, as defined herein. When a region of complementarity is not perfectly complementary to a target sequence, mismatches can be in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5' or 3' end of the iRNA. In certain embodiments, a double-stranded RNA agent of the invention contains nucleotide mismatches in the antisense strand. In certain embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the target mRNA, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In certain embodiments, the antisense strand of a double-stranded RNA agent of the invention contains four or fewer mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In certain embodiments, a double-stranded RNA agent of the invention contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of a double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA agent. In other embodiments, the nucleotide mismatch is, for example, the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0114] Thus, the RNAi agents described herein can contain one or more mismatches with the target sequence. In certain embodiments, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In certain embodiments, the RNAi agents described herein contain two or fewer mismatches. In certain embodiments, the RNAi agents described herein contain one or fewer mismatches. In certain embodiments, the RNAi agents described herein contain zero mismatches. In certain embodiments, if the antisense strand of an RNAi agent contains a mismatch with the target sequence, the mismatch can be limited to within the last five nucleotides from either the 5' or 3' end of the complementary region, as appropriate. For example, for a 23-nucleotide RNAi agent, the strand complementary to a region of the C3 gene, in such embodiments, generally does not contain any mismatches in the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an RNAi agent containing a mismatch in the target sequence is effective in inhibiting expression of the C3 gene. It is important to consider the effectiveness of mismatched RNAi agents in inhibiting expression of the C3 gene, especially if the particular complementary region in the C3 gene is known to have polymorphic sequence variation within the population.

[0115] As used herein, the terms "sense strand" or "passenger strand" refer to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0116] As used herein, "substantially all of the nucleotides are modified" means that a majority, but not all, of the nucleotides are modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0117] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises 3 bases at either end and immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises 2 bases at either end and immediately adjacent to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0118] As used herein, and unless otherwise specified, the term "complementarity," when referring to a first nucleotide sequence and a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions to form a duplex structure, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, and stringent conditions may be, for example, 400 mM N a Cl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in an organism, can be applied. One skilled in the art can determine the set of conditions that is most suitable for testing the complementarity of two sequences, depending on the final application of the hybridized nucleotides.

[0119] Complementary sequences within the iRNAs, e.g., dsRNAs, described herein include base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary or may form one or more, but generally not more than 5, 4, 3, or 2, mismatched base pairs upon hybridization for a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most appropriate for the end application, e.g., inhibiting gene expression via the RISC pathway. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches in determining complementarity. For example, if one oligonucleotide is 21 nucleotides in length and the other oligonucleotide is 23 nucleotides in length, and the longer oligonucleotide contains a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, then for purposes herein, this can still be referred to as "perfectly complementary."

[0120] As used herein, "complementary" sequences may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed from unnatural and modified nucleotides, so long as they meet the above requirements for hybridization ability. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0121] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matching between the sense and antisense strands of a dsRNA or between the antisense strand of a double-stranded RNA agent and a target sequence, as understood from the context in which they are used.

[0122] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding the complement component C3 gene). For example, a polynucleotide is complementary to at least a portion of a complement component C3 mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the complement component C3 gene.

[0123] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target complement component C3 sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target complement component C3 sequence, and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to the nucleotide sequence of any of SEQ ID NOs: 1-4 or the equivalent region of a fragment of any of SEQ ID NOs: 1-4.

[0124] In certain embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of a target complement component C3 sequence, including nucleotides 475-497, 487-509, 490-512, 491-513, 705-727, 809-831, 813-835, 1147-1169, 1437-1459, 1439-1461, 1447-1469, 2596-2618, 2634-2656, 3012-3034, 3334-3356, 3611-3633, 3614-3636, 3622-3655, 3809-3831, 3846-3868, 3847-3856, 3857-3859, 3861-3862, 3863-3864, 3865-3866, 3867-3868, 3870-3871, 3872-3873, 3874-3875, 3876-3878, 3879-3880, 3881-3882, 3883-3884, 3885-3886, 3887-3888, 3889-3890, 3891-3892, 3892-3893, 4893-4894, 4894-4895, 4895-4896, 4896-4897, 4897-4900, 4898-4901, 4899-4902 The fragment comprises a contiguous nucleotide sequence that is at least 80% complementary, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary over its entire length to a fragment of SEQ ID NO: 1 selected from the group consisting of: 3869, 3920-3942, 4047-4069, 4061-4083, 4156-4178, 4157-4177, 4162-4184, 4178-4200, 4226-4248, 4369-4391, 4392-4414, 4521-4543, 4522-4544, 4523-4545, and 5012-5034.

[0125] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target complement component C3 sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary over its entire length to a fragment of SEQ ID NO:1 selected from the group consisting of nucleotides 705-727, 809-831 or 2634-2656 of SEQ ID NO:1. In certain embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a fragment of the target complement component C3 sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary over its entire length to a fragment of SEQ ID NO:1 from nucleotides 2634 to 2656.

[0126] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target C3 sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% complementary over its entire length to any of the sense strand nucleotide sequences in any of Tables 2-7, 15, 18, 20-23, 30 and 31 or a fragment of any of the sense strand nucleotide sequences in any of Tables 2-7, 15, 18, 20-23, 30 and 31.

[0127] In certain embodiments, the RNAi agents of the present invention comprise a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is the same as a target C3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% complementary over its entire length to the nucleotide sequence of SEQ ID NOs: 5-8 or an equivalent region of a fragment of any of SEQ ID NOs: 5-8.

[0128] In certain embodiments, an iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is complementary to a target complement component C3 sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to any of the antisense strand nucleotide sequences of any of Tables 2-7, 15, 18, 20-23, 30, and 31, or a fragment of any of the antisense strand nucleotide sequences of any of Tables 2-7, 15, 18, 20-23, 30, and 31.

[0129] In some embodiments, the sense and antisense strands are duplexed with one of the following: AD-565541.2, AD-564742, AD-567304, AD-568978, AD-569164, AD-569272.2, AD-569765.2, AD-564730.2, AD-567315, AD-564745.2, AD-571715.2, AD-570714, AD-571826, AD-572041.2, AD-572039.2, AD-572387, AD-568586.2, AD-566837.2, AD-566444.2, AD-567700.2, AD -567814.2, AD-568003.2, AD-569164.2, AD-569763.2, AD-565281.2, AD-571539.2, AD-572389.2, AD-567315.2, AD-571752.2, AD-568026.2, AD-571298, AD-572110.2, AD-572062.2, AD-572388.2, AD-572040.2, AD-567713.2, AD-567521.2, AD-567066.2, AD-1181519, AD-569268 or AD-570714.

[0130] In some embodiments, the sense and antisense strands are selected from the duplex AD-1181519, AD-569268, or AD-570714. In some embodiments, the duplex is AD-570714.

[0131] Generally, "iRNA" comprises chemically modified ribonucleotides. Such modifications include all types of modifications disclosed herein or known in the art. Any such modifications used in dsRNA molecules are included in "iRNA" for purposes of this specification and claims.

[0132] In some embodiments of the present invention, the agent used in the methods and compositions of the present invention is a single-stranded antisense oligonucleotide molecule that inhibits target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing with the mRNA and physically interfering with the translation machinery (Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense oligonucleotide molecule can be about 14 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can contain a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of any of the antisense sequences described herein.

[0133] As used herein, the term "contacting an iRNA with a cell" (wherein the iRNA is, for example, a dsRNA) includes contacting the cell by any possible means. Contacting an iRNA with a cell includes contacting the iRNA with a cell in vitro or contacting the iRNA with a cell in vivo. Contacting can be performed directly or indirectly. Thus, for example, the iRNA can be brought into physical contact with the cell by the individual performing the method, or the iRNA can be placed in a condition that allows or will cause subsequent contact with the cell.

[0134] In vitro cell contact can be carried out, for example, by incubating cells with iRNA.In vivo cell contact can be carried out, for example, by injecting iRNA into the tissue where cells are located or nearby, or by injecting iRNA into other areas, for example, bloodstream or subcutaneous space, so that the iRNA will then reach the tissue where the agent is to contact cells.For example, iRNA can contain or be bound to a ligand, such as GalNAc, that directs iRNA to the target site, for example, liver.Combined in vitro and in vivo contact methods are also possible.For example, cells can be contacted with iRNA in vitro, and then transplanted into a subject.

[0135] In certain embodiments, contacting a cell with an iRNA includes "introducing" or "delivering" an iRNA to a cell by promoting or effecting uptake or absorption into the cell. Absorption or uptake of the iRNA can occur via unassisted diffusion or active cellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below or known in the art.

[0136] The term "lipid nanoparticle" or "LNP" refers to a vesicle containing a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid into which the iRNA is transcribed. LNPs are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0137] As used herein, a "subject" refers to an animal, such as a mammal, including a primate (e.g., a human, a non-human primate, e.g., a monkey or chimpanzee), a non-primate (e.g., a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird, that expresses a target gene endogenously or heterologously. In some embodiments, the subject is a human, such as a human being treated or evaluated for a disease or disorder that would benefit from reduced expression of complement component C3, as described herein; a human being at risk for a disease or disorder that would benefit from reduced C3 expression; a human being with a disease or disorder that would benefit from reduced C3 expression; or a human being treated for a disease or disorder that would benefit from reduced expression of complement component C3. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In some embodiments, the subject is an adult subject. In other embodiments, the subject is a pediatric subject.

[0138] As used herein, the term "treatment" or "treating" refers to a beneficial or desired result in a subject, such as reducing at least one sign or symptom of a complement component C3-associated disorder, e.g., hemolysis. Treatment also includes reducing one or more signs or symptoms associated with unwanted complement component C3 expression, e.g., hemolysis; reducing or stabilizing the degree of unwanted complement component C3 activation; or ameliorating or alleviating or stabilizing unwanted complement component C3 activation. "Treatment" can also mean prolonging survival compared to life expectancy in the absence of treatment.

[0139] The term "reduction" in the context of the level of complement component C3 gene expression or complement component C3 protein production in a subject, or a disease marker or symptom, refers to a statistically significant decrease in such level. The decrease can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or below the level of detection, for example, for detection methods in relevant cells or tissues, e.g., liver cells, or other subject samples, e.g., blood or serum derived therefrom, urine.

[0140] As used herein, "prevention" or "preventing" includes preventing a disease or disorder that would benefit from reduced expression of the complement component C3 gene or production of complement component C3 protein, e.g., in a subject predisposed to a complement component C3-related disorder due to, e.g., aging, genetic factors, hormonal changes, dietary habits, and a sedentary lifestyle. In certain embodiments, the disease or disorder is a symptom of unwanted C3 activation or stabilization, e.g., hemolysis. For example, the likelihood of developing hemolysis is reduced, e.g., an individual with one or more risk factors for hemolysis does not develop hemolysis, or the severity of hemolysis that develops is reduced, compared to a population with the same risk factors and not receiving a treatment described herein. Non-development of a complement component C3-related disorder, e.g., hemolysis, or a delay in the onset of hemolysis by several months or years is considered effective prevention. Prevention may require one or more administrations of an iRNA agent.

[0141] As used herein, the term "complement component C3-related disease" or "C3-related disease" refers to a disease or disorder that would benefit from reduced expression of complement component C3. Non-limiting examples of complement component C3-related diseases include cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

[0142] A "therapeutically effective amount" or a "prophylactically effective amount" also includes that amount of an RNAi agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNAs used in the methods of the invention can be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0143] The term "pharmaceutically acceptable" is used to refer to compounds, substances, compositions or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0144] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, additive, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium, or zinc, or stearic acid), or solvent encapsulating material, involved in the transport or transfer of a compound from one organ or part of the body to another organ or part of the body. The carrier must be "acceptable" in that it is compatible with the other ingredients of the formulation and not harmful to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0145] As used herein, the term "sample" includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues within a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs, or localized areas. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within these organs. In some embodiments, samples can be derived from the liver (e.g., the entire liver or a section of the liver, or certain cells within the liver, such as hepatocytes). In some embodiments, a "sample derived from a subject" can refer to urine obtained from a subject. A "sample derived from a subject" can refer to blood or blood-derived serum or plasma from a subject.

[0146] II. iRNA of the Invention The present invention provides iRNAs that inhibit expression of the complement component C3 gene. In a preferred embodiment, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits expression of the complement component C3 gene in cells within a subject, e.g., a mammal, such as a human, susceptible to a complement component C3-associated disorder, e.g., hemolysis. The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed by expression of the complement component C3 gene. The complementary region is approximately 19-30 nucleotides in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length). Upon contact with cells expressing the complement component C3 gene, the iRNA inhibits expression of the complement component C3 gene (e.g., human, primate, non-primate, or rat complement component C3 gene) by at least about 50%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, e.g., using Western blotting or flow cytometry techniques. In a preferred embodiment, inhibition of expression is determined by the qPCR method provided in the Examples, particularly Example 2, at a 10 nM concentration of siRNA in the appropriate biological cell line provided therein. In a preferred embodiment, inhibition of in vivo expression is determined by knockdown of the human gene at the nadir of RNA expression, for example, when administered at a single dose, e.g., 3 mg / kg, in rodents expressing the human gene, e.g., mice expressing the human target gene or AAV-infected mice. RNA expression in the liver is determined using the PCR method provided in Example 2.

[0147] dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure under the conditions that dsRNA is used.One strand (antisense strand) of dsRNA comprises a complementary region that is substantially complementary to target sequence and generally completely complementary.Target sequence can be derived from the sequence of mRNA formed during the expression of complement component C3 gene.The other strand (sense strand) comprises a region that is complementary to antisense strand, so that when combined under appropriate conditions, the two strands hybridize to form a duplex structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, instead of being in separate oligonucleotides.

[0148] Generally, the duplex structure is 19-30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19-30 nucleotides in length.

[0149] In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length or about 25 to about 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21 to 23 nucleotides can serve as a substrate for the Dicer enzyme. As will also be appreciated by those skilled in the art, the region of RNA targeting for cleavage is most often a portion of a large RNA molecule, often an mRNA molecule. Where appropriate, a "portion" of the mRNA target is a continuous sequence of the mRNA target long enough to be a substrate for RNAi-specific cleavage (i.e., cleavage via the RISC pathway).

[0150] Those skilled in the art will recognize that a duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in certain embodiments, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA, as long as it is processed as a functional duplex of, for example, 15-30 base pairs, targeting the desired RNA for cleavage. Thus, those skilled in the art will recognize that, in certain embodiments, miRNA is a dsRNA. In other embodiments, the dsRNA is not a naturally occurring miRNA. In other embodiments, an iRNA agent useful for targeting complement component C3 gene expression is not produced in a target cell by cleavage of a large dsRNA.

[0151] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have superior inhibitory properties compared to blunt-ended counterparts. The nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. The overhang may be in the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be at the 5'-end, the 3'-end, or both ends of the antisense or sense strand of the dsRNA.

[0152] dsRNA can be synthesized by standard methods known in the art.Double-stranded RNAi compounds of the present invention can be prepared by a two-step method.First, prepare each strand of double-stranded RNA molecules separately.Then, anneal the component strands.The individual strands of siRNA compounds can be prepared using liquid phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily produce oligonucleotide strands that contain unnatural or modified nucleotides.Similarly, single-stranded oligonucleotides of the present invention can be prepared using liquid phase or solid phase organic synthesis or both.

[0153] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences: a sense sequence and an antisense sequence. The sense strand is selected from the group consisting of the sequences provided in any of Tables 2-7, 15, 18, 20-23, 30, or 31, and the corresponding antisense strand of the sense strand is selected from the group consisting of the sequences provided in any of Tables 2-7, 15, 18, 20-23, 30, or 31. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of an mRNA gene produced upon expression of the complement component C3 gene. That is, in this embodiment, the dsRNA comprises two oligonucleotides, one oligonucleotide being listed as the sense strand in any of Tables 2-7, 15, 18, 20-23, 30, or 31, and a second oligonucleotide being listed as the corresponding antisense strand of the sense strand in any of Tables 2-7, 15, 18, 20-23, 30, or 31. In some embodiments, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides, hi other embodiments, the substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.In some embodiments, the sense strand or the antisense strand is a duplex AD-565541.2, AD-564742, AD-567304, AD-568978, AD-569164, AD-569272.2, AD-569765.2, AD-564730.2, AD-567315, AD-564745.2, AD-571715.2, AD-570714, AD-571826, AD-572041.2, AD-572039.2, AD-572387, AD-568586.2, AD-566837.2, AD-566444.2, AD-567700.2, AD-567814 .2, AD-568003.2, AD-569164.2, AD-569763.2, AD-565281.2, AD-571539.2, AD-572389.2, AD-567315.2, AD-571752.2, AD-568026.2, AD-571298, AD-572110.2, AD-572062.2, AD-572388.2, AD-572040.2, AD-567713.2, AD-567521.2, AD-567066.2, AD-1181519, AD-569268 or AD-570714. In other embodiments, the sense or antisense strand is selected from the sense or antisense strand of any of the duplexes AD-1181519, AD-569268, or AD-570714. In certain embodiments, the duplex is AD-570714.

[0154] Although the sequences in Tables 2, 4, 6, 20, 22, and 30 are not described as modified or conjugated sequences, the iRNAs of the invention, e.g., dsRNAs of the invention, can comprise any sequence set forth in any of Tables 3, 5, 7, 15, 18, 21, 23, or 31, unmodified, unconjugated, or modified or conjugated differently than described herein. In other words, the invention includes the dsRNAs of Tables 2-7, 15, 18, 20-23, 30, or 31, unmodified, unconjugated, modified, or conjugated as described herein.

[0155] Those skilled in the art will appreciate that dsRNAs having a duplex structure of approximately 20-23 base pairs, e.g., 21 base pairs, are considered to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above embodiments, the dsRNA described herein can comprise at least one strand a minimum of 21 nucleotides in length, according to the oligonucleotide sequences provided in any of Tables 2-7, 15, 18, 20-23, 30, or 31. It is reasonably expected that shorter duplexes having any sequence from any of Tables 2-7, 15, 18, 20-23, 30, or 31 minus only a few nucleotides at one or both ends will be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having at least 19, 20, or more contiguous nucleotide sequences from any of the sequences in any of Tables 2-7, 15, 18, 20-23, 30, or 31 that inhibit complement component C3 gene expression by no more than about 5%, 10%, 15%, 20%, 25%, or 30% less than dsRNAs containing the entire sequence are contemplated within the scope of the present invention.

[0156] Additionally, the RNAs provided in Tables 2-7, 15, 18, 20-23, 30, or 31 identify sites in the complement component C3 transcript that are susceptible to RISC-mediated cleavage. Thus, the present invention further relates to iRNAs that target one of these sites. As used herein, an iRNA is said to be targeted within a specific site in an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within this specific site. Such iRNAs generally comprise at least about 19 contiguous nucleotides from any of the sequences provided in any of Tables 2-7, 15, 18, 20-23, 30, or 31 linked to an additional nucleotide sequence taken from a region contiguous with the selected sequence in the complement component C3 gene.

[0157] III. Modified iRNAs of the Invention In some embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is unmodified, e.g., does not contain any chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of the iRNA of the present invention, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In some embodiments of the present invention, substantially all of the nucleotides of the iRNA of the present invention are modified. In other embodiments of the present invention, all of the nucleotides of the iRNA or substantially all of the nucleotides of the iRNA are modified. That is, no more than 5, 4, 3, 2, or 1 unmodified nucleotide is present in the strand of the iRNA.

[0158] Nucleic acids of the present invention can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA (incorporated herein by reference). Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, DNA nucleotides, reverse linkage, etc.); base modifications, such as substitutions of stabilizing bases, destabilizing bases, or bases that base-pair with an expanded repertoire of partners, base removal (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; or backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. RNAs with modified backbones include, inter alia, those containing a phosphorus atom in the backbone. For the purposes of this specification and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In certain embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0159] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In some embodiments, the dsRNA agent of the present invention is in sodium salt form. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions exist in the agent as counterions of substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.The agent in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterions comprises no more than 5, 4, 3, 2 or 1 phosphodiester and / or phosphorothioate linkages that do not have sodium counterions.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions exist in the agent as counterions of all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0160] Representative U.S. patents that teach the above phosphorus-containing linkages include U.S. Patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5,587,361;5,625, 050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464 (the entire contents of each of which are incorporated herein by reference).

[0161] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0162] Representative U.S. patents that teach the preparation of the above oligonucleosides include U.S. Patents 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,677,437; and 5,677,439 (the entire contents of each of which are incorporated herein by reference).

[0163] Suitable RNA mimics, in which both the sugar and internucleoside linkages, i.e., the backbone, of the nucleotide units are replaced with novel groups, are considered for use in the iRNAs provided herein. The base units are maintained for hybridization with appropriate nucleic acid target compounds. Such oligomeric compounds, which have been shown to have excellent hybridization properties with RNA mimics, are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262 (the entire contents of each of which are incorporated herein by reference). Further PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0164] Certain embodiments of the present invention include oligonucleosides having phosphorothioate and heteroatom backbones, and in particular, RNAs having --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--) of U.S. Patent 5,489,677 and amide backbones of U.S. Patent 5,602,240. In certain embodiments, the RNAs described herein have morpholino backbone structures of U.S. Patent 5,034,506.

[0165] Modified RNAs can contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein, can 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 (wherein alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl). Examples of suitable modifications include O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) nCH3)]2, where n and m are 1 to about 10. In other embodiments, the dsRNA can include one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA dissociating group, a reporter group, an intercalator, a group that improves the pharmacokinetic properties of an iRNA or a group that improves the pharmacodynamic properties of an iRNA, and other substituents with similar properties. In certain embodiments, the modification comprises 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications are the O(CH)ON(CH) group, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH-O-CH-N(CH). Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers of these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0166] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly the sugar of the 3'-terminal nucleotide or the 3' position of a 2'-5'-linked dsRNA and the 5' position of a 5'-terminal nucleotide. iRNAs can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include U.S. Patents 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591 ,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, portions of which are commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0167] iRNAs can also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, and 5-uracil. (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Patent 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L. ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y. S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds described in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and represent a further base substitution, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0168] Representative U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases, as well as other modified nucleobases, include U.S. Patents 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596 ,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088 (the entire contents of each of which are incorporated herein by reference).

[0169] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0170] In certain embodiments, the RNA of an iRNA can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified with a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in certain embodiments, an agent of the present invention can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-endo structural conformation. The addition of a locking nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides used in the polynucleotide of the present invention include, but are not limited to, nucleosides that contain a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the antisense polynucleotide agent of the present invention contains one or more bicyclic nucleosides that contain a 4'-2' bridge.Examples of such 4'-2' bridged bicyclic nucleosides include 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; e.g., U.S. Patent 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; e.g., U.S. Patent 8,222,222). 78,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0171] Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include the following: U.S. Patents 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,422 7,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US2008 / 0039618; and US2009 / 0012281 (the entire contents of each of which are incorporated herein by reference).

[0172] Any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0173] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In some embodiments, the constrained ethyl nucleotide has an S configuration, referred to herein as an "S-cEt."

[0174] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable structure and increases hybridization affinity to mRNA. The linker is long enough to place the oxygen in the optimal position for stability and affinity without significantly distorting the ribose ring.

[0175] Representative publications that teach the preparation of some of the above CRNs include, but are not limited to, U.S. Patent Publication 2013 / 0190383; and PCT Publication WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0176] In some embodiments, the iRNA of the present invention includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are non-locked acyclic nucleic acids in which any of the sugar linkages has been removed to form an unlocked "sugar" residue. As an example, UNAs also include monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, both of which are incorporated herein by reference).

[0177] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publications 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0178] Potential stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in PCT Publication WO2011 / 005861.

[0179] Other modifications of the nucleotides of the iRNAs of the invention include 5' phosphates or 5' phosphate mimics, such as 5' terminal phosphates or phosphate mimics, on the antisense strand of the iRNA. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication 2012 / 0157511, the contents of which are incorporated herein by reference in their entirety.

[0180] A. Modified iRNAs Containing Motifs of the Invention In some embodiments of the present invention, the double-stranded RNA agent of the present invention includes agents having chemical modifications, for example, as disclosed in WO2013 / 075035 (the entire contents of which are incorporated herein by reference). WO2013 / 075035 provides motifs of three identical modifications in the sense or antisense strand of the dsRNAi agent, particularly in three consecutive nucleotides at or near the cleavage site. In some embodiments, the sense and antisense strands of the dsRNAi agent may be completely modified otherwise. The introduction of these motifs disrupts the modification pattern, if any, of the sense or antisense strand. The dsRNAi agent may optionally be conjugated with a GalNAc derivative ligand, for example, on the sense strand.

[0181] More specifically, gene silencing activity of a dsRNAi agent was observed if the sense and antisense strands of the double-stranded RNA agent were fully modified to have one or more motifs of three identical modifications of three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0182] Thus, the present invention provides double-stranded RNA agents capable of inhibiting expression of a target gene (i.e., the complement component C3 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0183] The sense strand and antisense strand generally form a duplex double-stranded RNA ("dsRNA"), also referred to herein as a "dsRNAi agent." The duplex region of a dsRNAi agent can be, for example, 27-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In other examples, the duplex region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0184] In certain embodiments, a dsRNAi agent can include one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can independently be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides in length. In certain embodiments, the overhang region can include the extended overhang region provided above. The overhang can be the result of one strand being longer than the other or the result of two strands of the same length being offset. The overhang can form a mismatch with the target mRNA or can be complementary to the targeted gene sequence or other sequence. The first and second strands can be joined by additional bases or other non-basic linkers, for example, to form a hairpin.

[0185] In some embodiments, the nucleotides in the overhang region of dsRNAi agent can each independently be modified or unmodified nucleotides, including but not limited to 2'-sugar modifications, such as 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo) and any combination thereof.For example, TT can be the overhang sequence at either end of either strand.The overhang can form mismatches with target mRNA or can be complementary to the targeted gene sequence or other sequences.

[0186] The 5'- or 3'-overhang of the sense strand, antisense strand or both strands of dsRNAi agent can be phosphorylated.In some embodiments, the overhang region has two nucleotides with phosphorothioate between them, and these two nucleotides can be the same or different.In some embodiments, the overhang is present at the 3'-end of the sense strand, antisense strand or both strands.In some embodiments, this 3'-overhang is present at the antisense strand.In some embodiments, this 3'-overhang is present at the sense strand.

[0187] dsRNAi agent may contain only one overhang, which can enhance the interference activity of RNAi without affecting overall stability.For example, the single-stranded overhang can be located at the 3' end of the sense strand or the 3' end of the antisense strand.RNAi can also contain a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa.Generally, the antisense strand of dsRNAi agent has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without wishing to be bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the 3' end overhang of the antisense strand are advantageous for the guide strand to be guided by the RISC process.

[0188] In certain embodiments, the dsRNAi agent is a 19-nucleotide double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 7, 8, and 9, from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0189] In other embodiments, the dsRNAi agent is a 20-nucleotide double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 8, 9, and 10, from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0190] In yet other embodiments, the dsRNAi agent is a 21-nucleotide double-ended bluntmer, wherein the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 9, 10, and 11, from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13, from the 5' end.

[0191] In some embodiments, dsRNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 9, 10 and 11 from the 5' end, and antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12 and 13 from the 5' end, wherein one end of RNAi agent is blunt, while the other end comprises two-nucleotide overhang.Preferably, two-nucleotide overhang is at the 3' end of antisense strand.

[0192] When two nucleotides overhang at the 3'-end of antisense strand, there can be two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.In some embodiments, the RNAi agent further has two phosphorothioate internucleotide bonds between the terminal three nucleotides at the 5'-end of sense strand and the 5'-end of antisense strand.In some embodiments, all nucleotides of the sense strand and antisense strand of dsRNAi agent, including the nucleotide that is part of the motif, are modified nucleotides.In some embodiments, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif.Optionally, the dsRNAi agent further comprises a ligand (preferably GalNAc3).

[0193] In some embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length, and comprises at least 8 ribonucleotides starting from positions 1 to 23 of the 5'-terminal nucleotide (position 1) of the first strand, and the antisense strand is 36 to 66 nucleotide residues in length, and comprises at least 8 ribonucleotides starting from the 3'-terminal nucleotide and paired with positions 1 to 23 of the sense strand, forming a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired from the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired from the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; wherein the 5'-end of the antisense strand comprises 10 to 30 consecutive nucleotides, which the sense strand is unpaired, thereby forming a 10-30 nucleotide single-stranded 5' overhang; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

[0194] In some embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, wherein the dsRNAi agent comprises a first strand having a length of at least 25 and a maximum of 29 nucleotides, and a second strand having a length of at most 30 nucleotides, and having at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, 11, 12, and 13, from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the 3' end of the second strand is 1-4 nucleotides longer than the first strand, wherein the duplex region of at least 25 nucleotides in length and the second strand are sufficiently complementary to the target RNA along at least 19 ribonucleotides of the second strand so that the RNAi agent reduces target gene expression when introduced into mammalian cells, wherein Dicer cleavage of the dsRNAi agent preferentially produces siRNA containing the 3' end of the second strand, thereby reducing the expression of the target gene in mammalian cells. Optionally, the dsRNAi agent further comprises a ligand.

[0195] In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications of three consecutive nucleotides, where one of the motifs occurs at the cleavage site of the sense strand.

[0196] In certain embodiments, the antisense strand of the dsRNAi agent may also contain at least one motif of three identical modifications of three consecutive nucleotides, where one of the motifs occurs at or near the site of cleavage in the antisense strand.

[0197] For dsRNAi agents having a duplex region 19-23 nucleotides in length, the cleavage sites of the antisense strand are generally approximately positions 10, 11, and 12 from the 5' end. Thus, the three identically modified motifs can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide at the 5' end of the antisense strand or from the first pair of nucleotides within the duplex region at the 5' end of the antisense strand. The cleavage sites of the antisense strand can also vary depending on the length of the duplex region of the dsRNAi agent from the 5' end.

[0198] The sense strand of dsRNAi agent can comprise at least one motif of three identical modifications of three consecutive nucleotides at the site of strand breakage; and the antisense strand can comprise at least one motif of three identical modifications of three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides of sense strand and one motif of three nucleotides of antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif of sense strand forms base pairs with at least one of the three nucleotides of the motif of antisense strand.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0199] In some embodiments, the sense strand of a dsRNAi agent may contain more than one motif of three identical modifications of three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand, and the other motif may be a wing modification. The term "wing modification" herein refers to a motif occurring in another part of the strand, separate from the motif at or near the cleavage site of the same strand. The wing modification may be adjacent to the first motif or separated by at least one or more nucleotides. If the motifs are immediately adjacent to each other, the chemistry of the motifs may be different from each other, and if the motifs are separated by one or more nucleotides, the chemistry may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0200] Like the sense strand, the antisense strand of a dsRNAi agent may contain more than one motif of three identical modifications of three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand breakage. The antisense strand may also contain one or more wing modifications in a similar alignment to the wing modifications that may be present in the sense strand.

[0201] In certain embodiments, wing modifications of the sense or antisense strand of a dsRNAi agent generally do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0202] In other embodiments, wing modifications of the sense or antisense strand of a dsRNAi agent generally do not include the first one or two terminal nucleotides within the duplex region at the 3' end, 5' end, or both ends of the strand.

[0203] When the sense and antisense strands of a dsRNAi agent each include at least one wing modification, the wing modifications are at the same end of the duplex region and can have an overlap of 1, 2, or 3 nucleotides.

[0204] When the sense and antisense strands of a dsRNAi agent each contain at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from each strand are at one end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; two modifications from each strand are at the other end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; and two modifications from each strand are on either side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0205] In some embodiments, all nucleotides in the sense strand and antisense strand of dsRNAi agent, including the nucleotide that is part of motif, can be modified.Each nucleotide can be modified with the same or different modifications, and this can include one or both of non-linked phosphate oxygen or one or more of linked phosphate oxygen modification;Modification of ribose sugar component, for example, the 2'-hydroxyl of ribose sugar;Complete replacement of phosphate moiety with " dephosphorylation " linker;Modification or replacement of naturally occurring base;And replacement or modification of ribose-phosphate backbone.

[0206] Because nucleic acids are subunits of a polymer, many modifications, such as modifications of bases or phosphate moieties or non-linked Os in phosphate moieties, occur at positions that are repeated within the nucleic acid. In some cases, modifications occur at all target positions in the nucleic acid, but in many cases this is not the case. For example, modifications can occur only at the 3'- or 5'-terminal positions, or at the terminal regions of the chain, such as the terminal nucleotide positions or the last 2, 3, 4, 5, or 10 nucleotides. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA or only in single-stranded regions of RNA. For example, phosphorothioate modifications of non-linked Os can occur only at one or both ends, or only in the terminal regions, such as the terminal nucleotide positions or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or only in double-stranded and single-stranded regions, especially the termini. The 5'- or 3'-end can be phosphorylated.

[0207] For example, to enhance stability, it may be possible to include specific bases in the overhang or to include modified nucleotides or nucleotide surrogates in the single-stranded overhang, for example, in the 5'- or 3'-overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3'- or 5'-overhang can be modified, for example, with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar with modifications known in the art, such as deoxyribonucleotides in place of the ribosugar of the nucleobase, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications, and modifications at the phosphate group, such as phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0208] In some embodiments, each residue of sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.Strands can contain more than one modification.In some embodiments, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0209] At least two different modifications are generally present in the sense and antisense strands. These two modifications may be 2'-O-methyl or 2'-fluoro modifications or others.

[0210] In one embodiment, N a or N bincludes alternating patterns of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, with each modification occurring at alternating nucleotides on one strand. The alternating nucleotides can be one at every other nucleotide, or one at every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of nucleotide modification, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0211] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of nucleotide modification, the alternation pattern, i.e., the modifications of the nucleotides per one, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0212] In some embodiments, the dsRNAi agent of the present invention has a modification pattern of the alternating motif of the sense strand that is shifted relative to the modification pattern of the alternating motif of the antisense strand. The shift can be such that the modified group of the nucleotide of the sense strand corresponds to the differently modified group of the nucleotide of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand of a dsRNA duplex in the heavy strand region, the alternating motif of the sense strand can start with "ABABAB" at the 5' to 3' of the strand, and the alternating motif of the antisense strand can start with "BABABA" at the 5' to 3' of the double strand. As another example, the alternating motif of the sense strand in the duplex region can start with "AABBAABB" at the 5' to 3' of the strand, and the alternating motif of the antisense strand can start with "BBAABBAA" at the 5' to 3' of the strand, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0213] In some embodiments, dsRNAi agent comprises the alternating motif pattern of 2'-O-methyl modification and 2'-F modification of sense strand first, and the alternating motif pattern of 2'-O-methyl modification and 2'-F modification of antisense strand first shifted, that is, the 2'-O-methyl modified nucleotide of sense strand base pair can be the 2'-F modified nucleotide of antisense strand, and vice versa.The 1st position of sense strand can start with 2'-F modification, and the 1st position of antisense strand can start with 2'-O-methyl modification.

[0214] The introduction of one or more motifs of three identical modifications in three consecutive nucleotides of sense strand or antisense strand interrupts the initial modification pattern present in sense strand or antisense strand.The interruption of the modification pattern of sense strand or antisense strand by the introduction of one or more motifs of three identical modifications in three consecutive nucleotides of sense strand or antisense strand can enhance the gene silencing activity against target gene.

[0215] In some embodiments, when a motif of three identical modifications of three consecutive nucleotides is introduced into either strand, the modifications of the nucleotides adjacent to the motif are different modifications from the modification of the motif. For example, the portion of the sequence containing the motif is designated "...N" a YYYN b ...", where "Y" represents a modification of a motif of three identical modifications of three consecutive nucleotides, and "N a " and "N b " represents a modification different from the modification of the Y present in the nucleotide adjacent to the motif "YYY", where N a and N b may be the same or different modifications. Alternatively, N a or N b may or may not be present when wing modifications are present.

[0216] The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur at any nucleotide at any position in the sense strand, the antisense strand, or both strands. For example, the internucleotide linkage modification may occur at every nucleotide in the sense strand or the antisense strand; each internucleotide linkage modification may occur in an alternating pattern in the sense strand or the antisense strand; or the sense strand or the antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications in the sense strand may be the same as or different from that in the antisense strand, and the alternating pattern of internucleotide linkage modifications in the sense strand may be shifted relative to the alternating pattern of internucleotide linkage modifications in the antisense strand. In some embodiments, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at the 5' end or the 3' end.

[0217] In some embodiments, the dsRNAi agent comprises phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region.For example, the overhang region can comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide bond between them.Internucleotide bond modification can also be such that the overhang nucleotide is bonded to the terminal pair of nucleotides in the double-stranded region.For example, at least 2, 3, 4 or all of the overhang nucleotides can be bonded via phosphorothioate or methylphosphonate internucleotide bond, and optionally, there can be additional phosphorothioate or methylphosphonate internucleotide bond connecting the overhang nucleotide and the pair of nucleotides adjacent to the overhang nucleotide.For example, there can be two phosphorothioate internucleotide bonds between at least the terminal three nucleotides, two of the three nucleotides can be overhang nucleotides, and the third can be the pair of nucleotides adjacent to the overhang nucleotide.These terminal three nucleotides can be the 3'-end of the antisense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, or the 5'-end of the antisense strand.

[0218] In some embodiments, the 2-nucleotide overhang is at the 3'-end of the antisense strand, and there are two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.Optionally, the dsRNAi agent can further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0219] In some embodiments, dsRNAi agents can contain mismatches with targets or combinations thereof within the duplex. Mismatches can occur in overhang regions or duplex regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, the free energy of association or dissociation of a particular pairing; the simplest approach is to test each paired base, but neighbor-to-neighbor or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C; G:U is preferred to G:C; and I:C is preferred to G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred to canonical (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred to canonical pairings.

[0220] In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or non-canonical pairings or pairings containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0221] In some embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from A, dA, dU, U and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0222] In other embodiments, the 3'-terminal nucleotide of the sense strand is deoxythymine (dT) or the 3'-terminal nucleotide of the antisense strand is deoxythymine (dT), e.g., there is a short sequence of deoxythymine nucleotides, e.g., two dT nucleotides, at the 3'-end of the sense strand, antisense strand, or both strands.

[0223] In some embodiments, the sense strand sequence is represented by Formula (I): [ka] [During the ceremony, i and j are each independently 0 or 1; p and q each independently represent 0 to 6; each N a represents an oligonucleotide sequence containing independently 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides; where N b and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent a motif of three identical modifications of one triplet of consecutive nucleotides. Preferably, all of YYY are 2'-F modified nucleotides.

[0224] In one embodiment, N a or N b includes alternating pattern modifications.

[0225] In some embodiments, YYY motif occurs at or near the cleavage site of sense strand.For example, when dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, YYY motif can occur at or near the cleavage site of sense strand (for example, can occur at position 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13), counting from the first nucleotide from the 5' end; or optionally, counting from the first pair of nucleotides in the double-stranded region from the 5' end.

[0226] In certain embodiments, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand may therefore be represented by the formula: [ka]

[0227] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0228] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0229] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence that includes 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0230] Each of X, Y and Z may be the same or different.

[0231] In other embodiments, i is 0 and j is 0, and the sense strand has the formula [ka] It can be represented by:

[0232] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0233] In one embodiment, the antisense strand sequence of the RNAi is represented by Formula (II): [ka] [During the ceremony, k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' represents an oligonucleotide sequence containing independently 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represents an overhanging nucleotide; where N b ' and Y' do not have the same modification; and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications of one triplet of three consecutive nucleotides. It can be represented by:

[0234] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0235] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a duplex region 17 to 23 nucleotides long, the Y'Y'Y' motif occurs at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end; or optionally, counting from the first pair of nucleotides in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, and 13.

[0236] In some embodiments, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0237] In some embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0238] The antisense strand can therefore be represented by the formula: [ka]

[0239] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0240] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0241] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0242] In other embodiments, k is 0, l is 0, and the antisense strand has the formula [ka] It can be represented by:

[0243] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0244] Each of X', Y' and Z' may be the same or different from each other.

[0245] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand can be independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0246] In certain embodiments, the sense strand of a dsRNAi agent may comprise a YYY motif occurring at positions 9, 10, and 11 of the strand, counting from the first nucleotide from the 5' end, or optionally, counting from the first paired nucleotide in the duplex region from the 5' end, when the duplex region is 21 nt; and Y represents a 2'-F modification. The sense strand may further comprise a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0247] In certain embodiments, the antisense strand may comprise a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, counting from the first nucleotide from the 5' end, or optionally, counting from the first paired nucleotide in the duplex region from the 5' end; and Y' represents a 2'-O-methyl modification. The antisense strand may further comprise an X'X'X' motif or a Z'Z'Z' motif as a wing modification at opposite ends of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0248] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0249] Thus, a dsRNAi agent for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the iRNA duplex may be represented by the formula (III): [ka] [During the ceremony, i, j, k, and l are each independently 0 or 1; p, p', q and q' are each independently 0 to 6; each N a and N a ’represents an oligonucleotide sequence containing independently 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence containing 0 to 10 modified nucleotides; where each n p ',n p , n q ' and n q each may be present or absent and independently represents an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications of one triplet of consecutive nucleotides. It is expressed by:

[0250] In some embodiments, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In other embodiments, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0251] Examples of combinations of sense and antisense strands that form iRNA duplexes include the following: [ka]

[0252] When the dsRNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0253] When the dsRNAi agent is represented by formula (IIIb), each N brepresents an oligonucleotide sequence containing, independently, 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0254] When the dsRNAi agent is represented by formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0255] When the dsRNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ represents an oligonucleotide sequence containing, independently, 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b and N b ’ Each of the independently comprises an alternating pattern of modifications.

[0256] In formulae (III), (IIIa), (IIIb), (IIIc) and (IIId), X, Y and Z may be the same or different from one another.

[0257] When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can be base-paired with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides are base-paired with the corresponding Y' nucleotide; or all three Y nucleotides are base-paired with the corresponding Y' nucleotide.

[0258] When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with the corresponding Z' nucleotide; or all three Z nucleotides are base-paired with the corresponding Z' nucleotide.

[0259] When a dsRNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can be base-paired with one of the X' nucleotides, or at least two of the X nucleotides are base-paired with the corresponding X' nucleotide; or all three X nucleotides are base-paired with the corresponding X' nucleotide.

[0260] In some embodiments, the modification of a Y nucleotide is different from the modification of a Y' nucleotide, the modification of a Z nucleotide is different from the modification of a Z' nucleotide, or the modification of an X nucleotide is different from the modification of an X' nucleotide.

[0261] In certain embodiments, when the dsRNAi agent is represented by Formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In other embodiments, when the RNAi agent is represented by formula (IIId), N a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate bond.a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker (described below). a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0262] In certain embodiments, when the dsRNAi agent is represented by Formula (IIIa), N a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate bonds, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0263] In some embodiments, the dsRNAi agent is a multimer comprising at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double strands are connected by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each of the double strands can target the same gene or two different genes; or each of the double strands can target two different target sites of the same gene.

[0264] In some embodiments, dsRNAi agent is a multimer comprising 3, 4, 5, 6 or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein double strands are connected by a linker.The linker can be cleavable or non-cleavable.Optionally, the multimer further comprises a ligand.Each double strand can target the same gene or two different genes; or each double strand can target two different target sites of the same gene.

[0265] In some embodiments, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) are linked to each other at one or both of 5'-end and 3'-end, and optionally conjugated with ligand.Each agent can target the same gene or two different genes; or each agent can target two different target sites of the same gene.

[0266] In certain embodiments, RNAi agents of the present invention may comprise nucleotides with a low number of 2'-fluoro modifications, for example, nucleotides with 10 or fewer 2'-fluoro modifications. For example, RNAi agents may comprise nucleotides with 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 2'-fluoro modifications. In a specific embodiment, RNAi agents of the present invention comprise nucleotides with 10 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications, in the sense strand and 6 nucleotides with 2'-fluoro modifications in the antisense strand. In another specific embodiment, RNAi agents of the present invention comprise nucleotides with 6 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications, in the sense strand and 2 nucleotides with 2'-fluoro modifications in the antisense strand.

[0267] In other embodiments, the RNAi agent of the present invention may comprise nucleotides with a relatively low number of 2'-fluoro modifications, for example, nucleotides with two or fewer 2'-fluoro modifications. For example, the RNAi agent may comprise nucleotides with two, one, or zero 2'-fluoro modifications. In a specific embodiment, the RNAi agent may comprise nucleotides with two 2'-fluoro modifications, for example, zero 2'-fluoro modifications in the sense strand and two 2'-fluoro modifications in the antisense strand.

[0268] Various publications describe multimeric iRNAs that can be used in the methods of the present invention, including WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, the contents of each of which are incorporated herein by reference in their entirety.

[0269] As described in further detail below, iRNAs comprising one or more carbohydrate moieties conjugated to the iRNA can optimize one or more properties of the iRNA. Often, the carbohydrate moiety is attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier, to which a carbohydrate ligand is attached. Ribonucleotide subunits in which the ribose sugar of the subunit has been so replaced are referred to herein as ribose-replacement modified subunits (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., one in which all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one in which one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0270] Ligands can be attached to polynucleotides via carriers. The carriers contain (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally to a bond available and suitable for incorporation into the backbone of the carrier, such as a phosphate or modified phosphate, e.g., the sulfur-containing backbone of a ribonucleic acid. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, such as a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier that is connected to the selected moiety. The moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier via an intervening tether. Thus, the cyclic carrier often contains a functional group, such as an amino group, or generally provides a bond suitable for incorporation or tethering into the ring of another chemical entity, e.g., a ligand.

[0271] The iRNA may be conjugated to the ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is a serinol backbone or a diethanolamine backbone.

[0272] In other embodiments of the invention, the iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent can be represented by formula (L): [ka]

[0273] In Formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In some embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In some embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In some embodiments, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0274] C1 is a thermolabile nucleotide located at the opposite site of the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is the position of the sense strand that pairs with positions 2-8 of the 5' end of the nucleotide antisense strand. In one example, C1 is at position 15 from the 5' end of the sense strand. The C1 nucleotide carries a thermolabile modification, which may include an abasic modification; a mismatch with the opposite nucleotide in the duplex; and a sugar modification such as a 2'-deoxy modification or an acyclic nucleotide, for example, an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In some embodiments, C1 is: i) a mismatch with the opposite nucleotide in the antisense strand; ii) [ka] an abasic modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In some embodiments, the thermolabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase of the mismatch pair is a 2'-deoxynucleobase. In one example, the thermolabilizing modification in C1 is GNA or [ka] is.

[0275] T1, T1', T2', and T3' are each independently a nucleotide containing a modification that provides the nucleotide with steric bulk that is equal to or less than the steric bulk of a 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Methods for determining the steric effect of a nucleotide modification are known to those skilled in the art. The modification may be at the 2' position of the ribose sugar of the nucleotide, or a modification to a non-ribose nucleotide, acyclic nucleotide, or backbone of the nucleotide that is similar to or equal to the 2' position of the ribose sugar, providing the nucleotide with steric bulk that is equal to or less than the steric bulk of the 2'-OMe modification. For example, T1, T1', T2', and T3' are each independently selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In some embodiments, T1 is DNA. In some embodiments, T1' is DNA, RNA, or LNA. In some embodiments, T2' is DNA or RNA. In some embodiments, T3' is DNA or RNA.

[0276] n 1 , n 3 and q 1 are independently 4 to 15 nucleotides in length.

[0277] n 5 , q 3 and q 7 are independently 1 to 6 nucleotides in length.

[0278] n 4 , q 2 and q 6 are independently 1 to 3 nucleotides in length; or 4 is 0.

[0279] q 5 are independently 0 to 10 nucleotides in length.

[0280] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0281] Or, n 4 is 0 to 3 nucleotides in length.

[0282] In some embodiments, n 4 can be 0. As an example, n 4 is 0 and q 2 and q 6 is 1. In another example, n 4 is 0 and q 2 and q 6 is 1, with two phosphorothioate internucleotide modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide modifications within positions 18-23 (counting from the 5' end of the antisense strand).

[0283] In some embodiments, n 4 , q 2 and q 6 is 1 each.

[0284] In some embodiments, n 2 , n 4 , q 2 , q 4 and q 6 is 1 each.

[0285] In one embodiment, C1 is a sequence in which the sense strand is 19 to 22 nucleotides in length and n 4 When C1 is 1, it is at positions 14 to 17 of the 5' end of the sense strand. In certain embodiments, C1 is at position 15 of the 5' end of the sense strand.

[0286] In one embodiment, T3' begins at position 2 from the 5' end of the antisense strand. In one example, T3' is position 2 from the 5' end of the antisense strand, and q 6 is equal to 1.

[0287] In one embodiment, T1' begins at position 14 from the 5' end of the antisense strand. In one example, T1' is at position 14 from the 5' end of the antisense strand, and q 2 is equal to 1.

[0288] In an exemplary embodiment, T3' begins at position 2 from the 5' end of the antisense strand, and T1' begins at position 14 from the 5' end of the antisense strand. 6 is equal to 1, T1' starts at position 14 from the 5' end of the antisense strand, and q 2 is equal to 1.

[0289] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0290] In one embodiment, T1' is position 14 from the 5' end of the antisense strand. In one embodiment, T1' is position 14 from the 5' end of the antisense strand, and q 2 is equal to 1, and modifications at the 2' position or at non-ribose, acyclic or positions in the backbone provide less steric bulk than 2'-OMe ribose.

[0291] In one embodiment, T3' is position 2 from the 5' end of the antisense strand. In one example, T3' is position 2 from the 5' end of the antisense strand, and q 6is equal to 1, and modifications at the 2' position or at non-ribose, acyclic or backbone positions provide less steric bulk than 2'-OMe ribose.

[0292] In one embodiment, T1 is a cleavage site for the sense strand. In one embodiment, T1 is a cleavage site for the sense strand, where the sense strand is 19-22 nucleotides in length and n 2 is 1, it is 11 from the 5' end of the sense strand. In an exemplary embodiment, T1 is 19-22 nucleotides in length and n 2 When the cleavage site is 1, it is at position 11 from the 5' end of the sense strand.

[0293] In one embodiment, T2' begins at position 6 from the 5' end of the antisense strand. In one example, T2' is between positions 6 and 10 from the 5' end of the antisense strand, and q 4 is 1.

[0294] In an exemplary embodiment, T1 has a sense strand of 19-22 nucleotides in length and n 2 is 1, the cleavage site of the sense strand, for example, position 11 from the 5' end of the sense strand; T1' is position 14 from the 5' end of the antisense strand, and q 2 is equal to 1, T1' modification is at the 2' position of the ribose sugar or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose; T2' is at positions 6-10 from the 5' end of the antisense strand, and q 4 is 1; and T3' is position 2 from the 5' end of the antisense strand, and q 6 is equal to 1, and the T3' modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides steric bulk below 2'-OMe ribose.

[0295] In one embodiment, T2' begins at position 8 from the 5' end of the antisense strand. In one example, T2' begins at position 8 from the 5' end of the antisense strand, and q 4 is 2.

[0296] In one embodiment, T2' begins at position 9 from the 5' end of the antisense strand. In one example, T2' is at position 9 from the 5' end of the antisense strand, and q 4 is 1.

[0297] In some embodiments, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0298] In some embodiments, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0299] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0300] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0301] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0302] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0303] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0304] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0305] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; optionally with at least two additional TTs at the 3' end of the antisense strand.

[0306] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; optionally having at least two additional TTs at the 3' end of the antisense strand; and having two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0307] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0308] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0309] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0310] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0311] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q6 is 1, B4' is 2'-F, and q 7 is 1.

[0312] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end of the antisense strand).

[0313] The RNAi agent may contain a phosphorus-containing group at the 5'-end of the sense or antisense strand, such as 5'-phosphate (5'-P), 5'-phosphorothioate (5'-PS), 5'-phosphorodithioate (5'-PS2), 5'-vinylphosphonate (5'-VP), 5'-methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl. [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be any of the 5'-E-VP isomers. [ka] , 5'-Z-VP isomer [ka] Or it may be a mixture thereof.

[0314] In certain embodiments, an RNAi agent comprises a phosphorus-containing group at the 5'-end of the sense strand. In certain embodiments, an RNAi agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0315] In certain embodiments, the RNAi agent comprises a 5'-P. In certain embodiments, the RNAi agent comprises a 5'-P in the antisense strand.

[0316] In certain embodiments, the RNAi agent comprises a 5'-PS. In certain embodiments, the RNAi agent comprises a 5'-PS on the antisense strand.

[0317] In some embodiments, the RNAi agent comprises 5'-VP. In some embodiments, the RNAi agent comprises 5'-VP in the antisense strand. In some embodiments, the RNAi agent comprises 5'-E-VP in the antisense strand. In some embodiments, the RNAi agent comprises 5'-Z-VP in the antisense strand.

[0318] In certain embodiments, an RNAi agent comprises a 5'-PS2. In certain embodiments, an RNAi agent comprises a 5'-PS2 in the antisense strand.

[0319] In some embodiments, the RNAi agent comprises a 5'-PS2. In some embodiments, the RNAi agent comprises 5'-deoxy-5'-C-malonyl in the antisense strand.

[0320] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0321] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0322] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0323] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS2.

[0324] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0325] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-P.

[0326] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-PS.

[0327] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0328] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2.

[0329] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0330] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0331] In some embodiments, B1 is 2'-OMe or 2'-F, and n1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also contains a 5'-PS.

[0332] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0333] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS2.

[0334] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0335] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also contains a 5'-P.

[0336] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also contains a 5'-PS.

[0337] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end). The RNAi agent also includes a 5'-VP. The 5'-VP can be 5'-E-VP, 5'-Z-VP, or a combination thereof.

[0338] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also includes a 5'-PS2.

[0339] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0340] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0341] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0342] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0343] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNAi RNA agent also contains a 5'-PS2.

[0344] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0345] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-P.

[0346] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-PS.

[0347] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0348] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2.

[0349] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0350] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0351] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0352] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0353] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS2.

[0354] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0355] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-P.

[0356] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also contains a 5'-PS.

[0357] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0358] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2.

[0359] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0360] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0361] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0362] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand.

[0363] In certain embodiments, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0364] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0365] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0366] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also includes a 5'-P and a targeting ligand. In some embodiments, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0367] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0368] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In some embodiments, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0369] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0370] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 (counting from the 5' end). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0371] In some embodiments, B1 is 2'-OMe or 2'-F, and n1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0372] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0373] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In some embodiments, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0374] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0375] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0376] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0377] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0378] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In some embodiments, the 5'-VP is at the 5' end of the antisense strand, and the targeting ligand is at the 3' end of the sense strand.

[0379] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS2 and a targeting ligand. In one embodiment, the 5'-PS2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0380] In some embodiments, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5' end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0381] In certain embodiments, the RNAi agents of the invention (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21 and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14-16, 18, and 20 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23 and 2'F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the dsRNA agent has a two-nucleotide overhang on the 3'-end of the antisense strand and a blunt end on the 5'-end of the antisense strand.

[0382] In other specific embodiments, the RNAi agents of the invention are (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0383] In other specific embodiments, the RNAi agents of the invention are (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2'-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23 and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0384] In other specific embodiments, the RNAi agents of the invention (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21 and 2'-F modifications at positions 7, 9, 11, 13, and 15; and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23 and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0385] In other specific embodiments, the RNAi agents of the invention are (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 9 and 12 to 21 and 2'-F modifications at positions 10 and 11; and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0386] In other specific embodiments, the RNAi agents of the invention (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, and 13 and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14-21; and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23 and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0387] In other specific embodiments, the RNAi agents of the invention (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19-21 and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23, 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and desoxynucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a 4-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0388] In other specific embodiments, the RNAi agents of the invention (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21 and 2'-F modifications at positions 7 and 9 to 11; and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23 and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0389] In other specific embodiments, the RNAi agents of the invention (a) (i) 21 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21 and 2'-F modifications at positions 7 and 9 to 11; and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23 and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 21 and 22, and between nucleotides 22 and 23 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0390] In other specific embodiments, the RNAi agents of the invention are (a) (i) 19 nucleotides in length; (ii) an ASGPR ligand attached to the 3' end, the ASGPR ligand comprising three GalNAc derivatives attached via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-4, 6, and 10-19 and 2'-F modifications at positions 5 and 7-9; and (iv) phosphorothioate internucleotide bonds between nucleotides 1 and 2 and between nucleotides 2 and 3 (counting from the 5' end) a sense strand having and (b) (i) 21 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21 and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide bonds between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 19 and 20, and between nucleotides 20 and 21 (counting from the 5' end) antisense strand having wherein the RNAi agent has a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0391] In certain embodiments, the iRNA used in the methods of the invention is an agent selected from the agents listed in any of Tables 2-7, 15, 18, 20-23, 30, and 31. The agent can further comprise a ligand.

[0392] III. iRNA Conjugates to Ligands Other modifications of the iRNA of the present invention include chemically linking the iRNA to one or more ligands, moieties, or conjugates that enhance, for example, cellular activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556).In other embodiments, the ligand is cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecanediol or undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-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), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0393] In some embodiments, the ligand changes the distribution, targeting, or duration of the incorporated iRNA agent. In preferred embodiments, the ligand enhances the affinity of a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cell or organ compartment, tissue, organ, or body region, compared to a species without such a ligand. Preferred ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0394] Ligands include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, for example, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) 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 polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0395] Ligand can also comprise targeting group, for example, cell or tissue targeting agent, for example, lectin, glycoprotein, lipid or protein, for example, antibody that binds to specific cell types such as kidney cells.Targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimic.In some embodiments, ligand is multivalent galactose, for example, N-acetyl-galactosamine.

[0396] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Cholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamins, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocycles of Eu 3+ complex), dinitrophenyl, HRP or AP.

[0397] Ligands can be proteins, such as glycoproteins or peptides, molecules with specific affinity for co-ligands, or antibodies, such as antibodies that bind to specific cell types, such as hepatocytes. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0398] The ligand can be a substance, e.g., a drug, that can increase cellular uptake of the iRNA agent, e.g., by disrupting cellular microtubules, microfilaments, or intermediate filaments, e.g., by disrupting the cytoskeleton of the cell. The drug can be, e.g., taxol, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinoid, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0399] In some embodiments, the ligands binding to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic agents, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Examples of PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamins, and biotin. Oligonucleotides containing multiple phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., PK-modulating ligands) for the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0400] The ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing pendant reactive functional groups, such as those derived from the description of linking molecules for oligonucleotides (described below). This reactive oligonucleotide can react directly with commercially available ligands, synthetic ligands bearing any of a variety of protecting groups, or ligands with attached linking moieties.

[0401] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by commercial vendors, including, for example, Applied Biosystems® (Foster City, Calif.). Any other method for such synthesis known in the art may additionally or alternatively be used. The use of similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives, is also known.

[0402] In the ligand-conjugated iRNA and ligand-molecule sequence-specific binding nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled in a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing a ligand molecule or non-nucleoside ligand-sequence component.

[0403] When using a nucleotide-conjugate precursor that already carries a linking moiety, the synthesis of the sequence-specific linked nucleoside is generally completed, and then a ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0404] A. Lipid Conjugates In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue, for example, non-renal target tissue in the body.For example, the target tissue can be the liver, including liver parenchymal cells.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can be used to (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport to target cell or cell membrane, or (c) regulate the binding to serum protein, for example, HSA.

[0405] Lipid-based ligand can be used to inhibit, for example, control, the binding of conjugate to target tissue.For example, the stronger the lipid or lipid-based ligand that binds to HSA, the less likely it is to be targeted to kidney, and therefore the less likely it is to be removed from body.The weaker the lipid or lipid-based ligand that binds to HSA, the less likely it can be used to target conjugate to kidney.

[0406] In one embodiment, the lipid-based ligand binds to HSA. Preferably, the conjugate binds to HSA with sufficient affinity such that it preferably distributes to non-renal tissues, but preferably not with such strong affinity that HSA-ligand binding cannot be reversed.

[0407] In other embodiments, the lipid-based ligand binds only weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0408] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, e.g., proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Examples of vitamins include vitamins A, E, and K. Other examples of vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, such as hepatocytes. Also included are HSA and low-density lipoprotein (LDL).

[0409] B. Cell-penetrating agents In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. Examples of agents are peptides such as tat or antennapedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and the use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and lipophobic phase.

[0410] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined tertiary structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0411] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety can contain a hydrophobic membrane translocating sequence (MTS). An example of a hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the sequence from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified in phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic tethered to a dsRNA agent by an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications to increase stability or direct structural properties. Any of the structural modifications described below can be utilized.

[0412] The RGD peptides used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptidomimetics can include D-amino acids and synthetic RGD mimics. In addition to RGD, other moieties that target integrin ligands can be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0413] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, cell-penetrating peptides can be bipartite amphipathic peptides such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0414] C. Carbohydrate Conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for compositions suitable for in vivo delivery and in vivo therapeutic applications of the nucleic acids described herein. As used herein, "carbohydrate" refers to a compound that is a carbohydrate itself, consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom attached to each carbon atom; or a compound that has as a moiety a carbohydrate moiety consisting of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 or greater (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0415] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.

[0416] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0417] In other embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In certain embodiments, the monosaccharide is [ka] and other N-acetylgalactosamines.

[0418] Other exemplary carbohydrate conjugates for use in the embodiments described herein include: [ka] and when one of X or Y is an oligonucleotide, the other is hydrogen.

[0419] In some embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker.

[0420] In certain embodiments, a double-stranded RNAi agent of the invention includes one or more GalNAc or GalNAc derivatives attached to an iRNA agent. The GalNAc may be attached to any nucleotide via a linker in the sense or antisense strand. a c can be attached to the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 3' end of the antisense strand. In some embodiments, GalNAc is attached to the 3' end of the sense strand, e.g., via a trivalent linker.

[0421] In other embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple linkers, e.g., monovalent linkers.

[0422] In certain embodiments, for example, when the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each other strand that form a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0423] In certain embodiments, the carbohydrate conjugate may further comprise one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0424] Additional carbohydrate conjugates and linkers suitable for use in the present invention include those disclosed in PCT Publications WO2014 / 179620 and WO2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0425] D. Linker In certain embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotides with a variety of linkers, which can be cleavable or non-cleavable.

[0426] The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound, e.g., covalently bonds two parts of a compound. A linker is generally a direct bond or an atom, such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a chain of atoms, including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylarylalkynyl,

[0033] The term "heteroarylalkyl," "alkenylheteroarylalkenyl," "alkenylheteroarylalkynyl," "alkynylheteroarylalkyl," "alkynylheteroarylalkenyl," "alkynylheteroarylalkynyl," "alkylheterocyclylalkyl," "alkylheterocyclylalkenyl," "alkylheterocyclylalkynyl," "alkenylheterocyclylalkyl," "alkenylheterocyclylalkenyl," "alkenylheterocyclylalkynyl," "alkynylheterocyclylalkyl," "alkynylheterocyclylalkenyl," "alkynylheterocyclylalkynyl," alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylenes may be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; wherein R is hydrogen, acyl, aliphatic, or substituted aliphatic.In some embodiments, the linker is about 1 to 24 atoms, 2 to 24 atoms, 3 to 24 atoms, 4 to 24 atoms, 5 to 24 atoms, 6 to 24 atoms, 6 to 18 atoms, 7 to 18 atoms, 8 to 18 atoms, 7 to 17 atoms, 8 to 17 atoms, 6 to 16 atoms, 7 to 17 atoms, or 8 to 16 atoms.

[0427] A cleavable linker is one that is sufficiently stable outside a cell, but cleaves upon entering a target cell to release the two moieties held together by the linker. In preferred embodiments, the cleavable linker is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least 100-fold faster inside the target cell or under first control conditions (which may, for example, be selected to mimic or represent intracellular conditions), compared to under the subject's blood or second control conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0428] Cleavable linking groups are sensitive to cleaving factors, such as pH, redox potential, or the presence of degradative molecules. Generally, cleaving factors are more prevalent or found at higher levels or activity within cells than in serum or blood. Examples of such degradative factors include redox agents that are selective for a particular substrate or do not have substrate specificity, such as oxidizing or reducing enzymes or reducing agents present in cells that can degrade redox-cleavable linking groups by reduction, such as mercaptans; esterases; endosomes or agents that can create an acidic environment, for example, a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0429] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand into the cell or a desired compartment of the cell.

[0430] The linker can comprise a cleavable linking group that can be cleaved by a specific enzyme.The type of cleavable linking group incorporated into the linker can depend on the target cell.For example, a liver targeting ligand can be linked to a cationic lipid via a linker that comprises an ester group.Hepatocytes are rich in esterase, and therefore the linker is cleaved more efficiently in hepatocytes than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0431] Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as hepatocytes and synoviocytes.

[0432] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group's ability to withstand cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition and a second condition, where the first is selected to exhibit cleavage in target cells and the second is selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0433] i. Redox-cleavable linking group In some embodiments, the cleavable linker is a redox-cleavable linker that is cleaved by reduction or oxidation. An example of a reductively cleavable linker is a disulfide linker (-SS-). The methods described herein can be used to determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent. For example, candidates can be evaluated by incubating them with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In one example, the candidate compound is cleaved at most about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0434] ii. Phosphate-based cleavable linkers In other embodiments, the cleavable linker comprises a phosphate-based cleavable linker. The phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that degrades phosphate groups in cells is an enzyme such as a phosphatase in cells. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0435] iii. Acid-cleavable linking group In other embodiments, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or by an agent such as an enzyme that can act as a general acid. In cells, specific low-pH organelles such as endosomes and lysosomes can provide an environment for cleavage of the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when the carbon is bonded to the oxygen of the ester (alkoxy group), it is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0436] iv. Ester-based linking groups In other embodiments, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved by enzymes in cells, such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0437] v. Peptide-based leaving groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved by enzymes, such as peptidases and proteases, in cells. Peptide-based cleavable linkers are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkylene. A peptide bond is a specific type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to produce peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0438] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to: [ka] [ka] (In the formula, when one of X or Y is an oligonucleotide, the other is hydrogen.)

[0439] In certain embodiments of the compositions and methods of the invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0440] In certain embodiments, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group consisting of the structure shown in any of formulas (XLV)-(XLVI). [ka] [During the ceremony, q 2A , q 2B , q 3A , q 3B , q 4A , q 4B , q 5A , q 5B and q 5C represents independently in each occurrence 0 to 20, where the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is independently at each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently at each occurrence absent, alkylene, or substituted alkylene, where one or more methylenes are O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C is independently in each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C is a ligand; i.e., independently in each occurrence, a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a is H or an amino acid side chain). Formula (XLIX) [ka] [In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative. Trivalently linked GalNAc derivatives such as are particularly useful for use in RNAi agents for target gene expression.

[0441] Examples of suitable divalent and trivalent branched linker group-linked GalNAc derivatives include, but are not limited to, the structures shown above as Formulas II, VII, XI, X, and XIII.

[0442] Representative U.S. patents that teach the preparation of RNA conjugates include U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136; 214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241, 5,391,723;5,416,203, 5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5,58 5,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022 (the entire contents of each of which are incorporated herein by reference).

[0443] It is not necessary for all positions in a compound to be uniformly modified, and in fact more than one of the above modifications may be incorporated in a single compound or at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0444] A "chimeric" iRNA compound or "chimera" in the context of the present invention is an iRNA compound, preferably a dsRNAi agent, that contains two or more chemically distinct regions, each comprising at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs generally contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA serves as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Therefore, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of iRNA inhibition of gene expression. As a result, comparable results can often be obtained with shorter iRNAs when using chimeric dsRNAs compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, by associated nucleic acid hybridization techniques known in the art.

[0445] In some cases, the RNA of an iRNA can be modified with a non-ligand group. Numerous non-ligand molecules have been conjugated to iRNAs to increase the activity, cellular distribution, or cellular uptake of the iRNA, and methods for performing such conjugation can be found in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating agent. The conjugation reaction can be performed with the RNA still bound to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC generally provides a pure conjugate.

[0446] IV. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, e.g., a cell in a subject, such as a human subject (e.g., a subject in need thereof, such as a subject susceptible to or diagnosed with a complement component C3-associated disorder, e.g., hemolysis), can be achieved in a number of different ways. For example, delivery can be performed by contacting the iRNA of the invention with a cell in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition containing the iRNA, e.g., dsRNA, to the subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that encode and direct expression of the iRNA. These options are further described below.

[0447] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider for delivery of iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. RNA interference has also been successfully delivered locally to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al. (2005) Gene Ther. 12:59-66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602). Modification of RNA or pharmaceutical carriers can also enable targeting of iRNA to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, systemic injection of iRNA against ApoB conjugated to a lipophilic cholesterol moiety into mice resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).

[0448] In another embodiment, iRNA is delivered using a drug delivery system such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of iRNA molecules (negatively charged) and also enhance their interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNA or induced to form vesicles or micelles that encapsulate iRNA (see, e.g., Kim SH, et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNA when administered systemically. Methods for preparing and administering cationic-iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, DR, et al (2003) J. Mol. Biol 327:761-766; Verma, UN, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN, et al (2003), supra), "solid nucleic acid lipid particles" (Zimmermann, TS, et al (2006) Nature 441:111-114), cardiolipin (Chien, PY, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487) and polyamidoamines (Tomalia, DA, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, iRNAs are complexed with cyclodextrins for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Patent 7,427,605, which is incorporated herein by reference in its entirety.

[0449] A. Vectors Encoding iRNAs of the Invention iRNA targeting the complement component C3 gene can be expressed from a transcription unit inserted into a DNA or RNA vector (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al., International Publication WO 00 / 22113; Conrad, International Publication WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (on the order of hours to weeks) or persistent (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. These 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 to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0450] Viral vector systems that can be utilized in the methods and compositions described herein 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 vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avian pox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenovirus. Replication-deficient viruses may also be advantageous. Various vectors may or may not integrate into the cellular genome. The constructs may contain viral sequences for transfection, if desired. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure expression of the iRNA in the target cell. Other aspects to consider in vectors and constructs are known in the art.

[0451] V. Pharmaceutical Compositions of the Invention The present invention also relates to pharmaceutical compositions and formulations comprising the iRNA of the present invention. In certain embodiments, provided herein are pharmaceutical compositions comprising the iRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising the iRNA are useful for preventing or treating complement component C3-associated disorders, such as hemolysis. Such pharmaceutical compositions are formulated according to the delivery method. One example is a composition formulated for systemic administration via parenteral delivery, for example, by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the present invention can be administered at a dose sufficient to inhibit expression of the complement component C3 gene.

[0452] In certain embodiments, the pharmaceutical compositions of the present invention are sterile. In other embodiments, the pharmaceutical compositions of the present invention are pyrogen-free.

[0453] The pharmaceutical compositions of the present invention can be administered at a dosage sufficient to inhibit expression of the complement component C3 gene. Generally, suitable doses of iRNAs of the present invention range from about 0.001 to about 200.0 mg / kg of recipient body weight / day, generally from about 1 to 50 mg / kg body weight / day. Generally, suitable doses of iRNAs of the present invention range from about 0.1 mg / kg to about 5.0 mg / kg, preferably from about 0.3 mg / kg to about 3.0 mg / kg. Repeated administration regimens can include administering a therapeutic dose of iRNA periodically, such as monthly, once every 3 to 6 months, or once a year. In certain embodiments, iRNAs are administered from about once a month to about once every 6 months.

[0454] After the initial treatment regimen, treatment may be administered less frequently. The duration of treatment may be determined based on the severity of the disease.

[0455] In other embodiments, the single administration of the pharmaceutical composition can be continuous, such that the dose is administered at 1-month, 2-month, 3-month, or 4-month intervals. In one embodiment of the present invention, the single administration of the pharmaceutical composition of the present invention is administered approximately once a month. In another embodiment of the present invention, the single administration of the pharmaceutical composition of the present invention is administered quarterly (i.e., approximately every 3 months). In another embodiment of the present invention, the single administration of the pharmaceutical composition of the present invention is administered twice a year (i.e., approximately once every 6 months).

[0456] Those of skill in the art will recognize that certain factors may affect the dosage and timing required to effectively treat a subject, including, but not limited to, mutations in the subject, previous treatments, the general health or age of the subject, and other coexisting diseases. Moreover, treatment of a subject with a prophylactically or therapeutically effective amount of a composition, as appropriate, can include a single treatment or a series of treatments.

[0457] iRNA can be delivered in a manner that targets specific tissues (e.g., liver cells).

[0458] The pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations.These compositions can be produced from a variety of components, including, but not limited to, pre-formulated liquids, self-emulsifying solids, and self-emulsifying semi-solids.Formulations include those that target the liver.

[0459] The pharmaceutical formulations of the present invention, which can be conveniently provided in unit dosage form, can be prepared by conventional techniques well known in the pharmaceutical industry. Such techniques include the step of combining the active ingredient with pharmaceutical carriers or excipients. Generally, the formulations are prepared by uniformly and intimately combining the active ingredient with liquid carriers.

[0460] A. Further Formulations i. Emulsion The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are generally heterogeneous systems of one liquid dispersed in another, typically in the form of droplets exceeding 0.1 μm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245). (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often two-phase systems containing two immiscible liquid phases intimately mixed with each other. Generally, emulsions can be of the water-in-oil (w / o) or oil-in-water (o / w) variety. When the aqueous phase is finely divided and dispersed as fine droplets in the bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as fine droplets in the bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion.Emulsions may contain additional components in addition to the active drug, which may exist as a dispersed phase and solution in the aqueous phase, oil phase, or as a separate phase. Pharmaceutical additives such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed. Pharmaceutical emulsions may also be multiple emulsions consisting of more than two phases, such as oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions. Such complex formulations often offer certain advantages over simple two-phase emulsions. A multiple emulsion in which individual oil droplets of an o / w emulsion encapsulate small water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets encapsulated in small water droplets stabilized in an oily continuous phase provides an o / w / o emulsion.

[0461] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase, and maintains this form through the emulsifier or viscosity of the formulation. Other means of emulsion stabilization include the use of emulsifiers, which can be incorporated into any phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0462] Synthetic surfactants, also known as surface active agents, have been found to be widely applicable to emulsion formulations and have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199).Surfactants are generally amphiphilic and comprise hydrophilic and hydrophobic moieties. The ratio of the hydrophilicity to the hydrophobicity of surfactant is called hydrophilic / lipophilic balance (HLB), and is a valuable tool for classifying and selecting surfactants in formulation preparation.Surfactants can be classified into various classes based on the nature of hydrophilic group, including nonionic, anionic, cationic and amphoteric (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0463] A wide variety of non-emulsifying substances are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0464] The application of emulsion formulations via the dermal, oral, and parenteral routes and methods for their preparation have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0465] ii. Microemulsions In some embodiments of the present invention, iRNA and nucleic acid compositions are formulated as microemulsions. Microemulsions can be defined as a system of water, oil, and amphiphiles that is a single optically isotropic and thermodynamically stable liquid solution (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Generally, microemulsions are systems that are prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally a medium-chain alcohol, to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically well-defined dispersions of two immiscible liquids stabilized by a thin interfacial layer of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).

[0466] iii. Particulates The iRNA of the present invention can be incorporated into particles, such as microparticles. Microparticles can be produced by spray drying, but can also be produced by other methods, including freeze-drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

[0467] iv. Penetration enhancers In some embodiments, the present invention uses various penetration enhancers to efficiently deliver nucleic acids, particularly iRNA, to animal skin.Most drugs exist in both ionized and non-ionized forms in solution.However, usually, only lipid-soluble or lipophilic drugs can easily pass through cell membranes.It has been discovered that non-lipophilic drugs can also pass through cell membranes if the membrane they are passing through is treated with penetration enhancers.In addition to aiding the diffusion of non-lipophilic drugs through cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0468] Penetration enhancers can be classified into one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above classes of penetration enhancers and their use in the manufacture of pharmaceutical compositions and drug delivery are well known in the art.

[0469] v. Additives In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivery of one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected to provide the desired bulk, viscosity, etc. when combined with the nucleic acid and other components of a pharmaceutical composition, depending on the intended method of administration. Such agents are well known in the art.

[0470] vi. Other Components The compositions of the present invention may further contain other adjuvant components commonly found in pharmaceutical compositions at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional substances useful for the physical formulation of various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, such substances should not excessively interfere with the biological activity of the components of the compositions of the present invention. The formulation can be stabilized, and if desired, can be mixed with adjuvants that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring, flavoring, or aromatic substances.

[0471] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also contain stabilizers.

[0472] In certain embodiments, pharmaceutical compositions of the invention can include (a) one or more iRNAs and (b) one or more agents that function by a non-iRNA mechanism and are useful in treating complement component C3-associated disorders, e.g., hemolysis.

[0473] The toxicity and prophylactic efficacy of such compounds can be assessed, for example, by LD 50 (a dose lethal to 50% of the population) and ED 50 The LD is a dose that is effective in 50% of a population and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine the dose that is prophylactically effective in 50% of a population. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD is the dose that is effective in 50% of a population. 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred.

[0474] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosages of the compositions described herein generally result in little or no toxicity and are within the ED 50 , preferably ED80 or ED 90 The dose may vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the prophylactically effective dose can be estimated initially from cell culture assays. Dosages are calculated in animal models, determined in cell culture, and then compared to the IC 50 A circulating plasma concentration range of the compound or, if appropriate, the polypeptide product of the target sequence can be achieved (e.g., a reduction in the concentration of the polypeptide) that includes an inhibitory level at or above the maximum concentration of the test compound (i.e., a test compound concentration that achieves half-maximal inhibition of pathogenesis). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0475] In addition to the above administrations, iRNAs of the invention can be administered in combination with other known agents used to prevent or treat complement component C3-associated disorders, such as hemolysis. In any event, the administering physician can adjust the amount and timing of iRNA administration based on the results observed using standard measures of efficacy known in the art or described herein.

[0476] VI. Methods of Inhibiting Complement Component C3 Expression The present invention also provides a method for inhibiting expression of the C3 gene in a cell, the method comprising contacting the cell with an RNAi agent, e.g., a double-stranded RNA agent, in an amount effective to inhibit expression of complement component C3 in the cell, thereby inhibiting expression of complement component C3 in the cell.

[0477] The contact between cells and RNAi agents, for example, double-stranded RNA agents, can be carried out in vitro or in vivo.The contact between cells and iRNA in vivo includes the contact between iRNA and cells or cell groups in a subject, for example, a human subject.A combined in vitro and in vivo cell contact method is also possible.As described above, the contact with cells can be direct or indirect.In addition, the contact with cells can be achieved through a targeting ligand, including any ligand described above or known in the art.In a preferred embodiment, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand, or any other ligand that directs RNAi agents to a target site.

[0478] As used herein, the term "inhibition" is used interchangeably with "reduction," "silencing," "downregulation," "suppression," and other similar terms, and includes any level of inhibition.

[0479] The term "inhibiting expression of complement component C3" is intended to refer to the inhibition of expression of any complement component C3 gene (e.g., mouse complement component C3 gene, rat complement component C3 gene, monkey complement component C3 gene, or human complement component C3 gene, etc.), as well as variants or mutants of the complement component C3 gene. Thus, the complement component C3 gene can be a wild-type complement component C3 gene, a mutant complement component C3 gene, or a transgenic complement component C3 gene in the context of a genetically engineered cell, cell population, or organism.

[0480] "Inhibition of complement component C3 gene expression" includes any level of inhibition of the complement component C3 gene, e.g., at least partial suppression of complement component C3 gene expression. Complement component C3 gene expression can be assessed based on the level or change in the level of any variable associated with complement component C3 gene expression, e.g., complement component C3 mRNA level or complement component C3 protein level. This level can be assessed in individual cells or groups of cells, including, for example, samples derived from a subject. It is understood that complement component C3 is predominantly expressed in the liver, but is also expressed in the brain, gallbladder, heart, and kidney, and is present in the circulation.

[0481] Inhibition can be assessed by a decrease in the absolute level of one or more variables associated with complement component C3 expression or a relative level compared to a control level, which can be any type of control level available in the art, such as a pre-administration baseline level or a level determined from similar subjects, cells, or samples that are untreated or treated with a control (e.g., a buffer-only control or an inactive agent control).

[0482] In some embodiments of the method of the present invention, the expression of complement component C3 gene can be inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or below the detection level of the assay.In a preferred embodiment, the expression of complement component C3 gene is inhibited by at least 70%.It is understood that it may be desirable to inhibit the expression of complement component C3 in some tissues, such as the liver, without significant expression inhibition in other tissues, such as the brain.In a preferred embodiment, the expression level is determined at a 10 nM siRNA concentration in an appropriate species-matched cell line using the assay method provided in Example 2.

[0483] In some embodiments, inhibition of in vivo expression is determined, for example, in rodents expressing a human gene, such as AAV-infected mice expressing a human target gene (i.e., complement component C3), by knockdown of the human gene at the nadir of RNA expression when administered at a single dose, e.g., 3 mg / kg. Knockdown of endogenous gene expression in a model animal system can also be determined, for example, after a single dose of 3 mg / kg at the nadir of RNA expression. Such a system is useful when the nucleic acid sequences of the human gene and the model animal gene are sufficiently similar so that the human iRNA provides effective knockdown of the model animal gene. RNA expression in the liver is determined using the PCR method provided in Example 2.

[0484] Inhibition of expression of the complement component C3 gene may be manifested by a decrease in the amount of mRNA expressed in a first cell or population of cells (such cells may be present, for example, in a sample derived from a subject) in which the complement component C3 gene is transcribed and which have been treated (e.g., by contacting one or more cells with an iRNA of the invention or by administering an iRNA of the invention to a subject in which the cells are or were present), compared to a second cell or population of cells substantially identical to the first cell or population of cells but which have not been so treated (control cells not treated with an iRNA or an iRNA targeting a gene of interest), such that expression of the complement component C3 gene is inhibited. In a preferred embodiment, inhibition is assessed using a 10 nM siRNA concentration in a species-matched cell line by the method provided in Example 2, and is calculated according to the following formula:

number

[0485] In other embodiments, inhibition of complement component C3 gene expression can be assessed in terms of a decrease in a parameter functionally related to complement component C3 gene expression, such as complement component C3 protein level, in the blood or serum from a subject. Complement component C3 gene silencing can be determined in any cell that expresses complement component C3 endogenously or heterologously from an expression construct and by any assay known in the art.

[0486] Inhibition of expression of complement component C3 protein may be manifested by a decrease in the level of complement component C3 protein expressed in a cell or group of cells or a subject sample (e.g., the level of the protein in a blood sample from the subject). As noted above, for inhibition of mRNA suppression, inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells or as a change in the level of protein in a subject sample, e.g., blood or serum derived therefrom.

[0487] The control cell, cell group or subject sample that can be used to evaluate the inhibition of the expression of complement component C3 gene includes the cell, cell group or subject sample that has not yet been contacted with the RNAi agent of the present invention.For example, the control cell, cell group or subject sample can be derived from an individual subject (e.g., human or animal subject) before the subject is treated with the RNAi agent, or from an appropriately matched population control.

[0488] The level of complement component C3 mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In some embodiments, the expression level of complement component C3 in a sample is determined by detecting a transcribed polynucleotide or a portion thereof, for example, the mRNA of the complement component C3 gene. RNA can be extracted, for example, by acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy TM RNA preparation kit (Qiagen (registered information) or PAXGENE) TM (PreAnalytix TM RNA can be extracted from cells using RNA extraction techniques, including the use of RNAi kits (Bio-Rad Laboratories, Inc., Switzerland). Exemplary assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis.

[0489] In one embodiment, the expression level of complement component C3 is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific complement component C3. Probes can be synthesized by those skilled in the art or derived from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0490] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to complement component C3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA through an agarose gel, and then transferring the mRNA from the gel to a membrane such as nitrocellulose. In another embodiment, the probe is immobilized on a solid surface and contacted with the mRNA, for example, in an Affymetrix gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in determining the level of complement component C3 mRNA.

[0491] Other methods for determining the expression level of complement component C3 in a sample include, for example, RT-PCR (Mullis, 1987, experimental embodiment shown in U.S. Patent 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., The process involves nucleic acid amplification of a sample's mRNA, for example, by reverse transcriptase (to prepare cDNA), using U.S. Patent No. 5,854,033 (U.S. Patent No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules if such molecules are present in very low numbers. In a specific embodiment of the present invention, the expression level of C3 is determined by quantitative fluorescent RT-PCR (i.e., TaqMan TMIn a preferred embodiment, expression levels are determined by the method provided in Example 2, for example, using a 10 nM siRNA concentration in a species-matched cell line.

[0492] The expression level of complement component C3 mRNA can be monitored using membrane blots (e.g., as used in hybridization analyses such as Northern, Southern, and dot analyses) or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid). See U.S. Patents 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining the expression level of complement component C3 can also involve the use of nucleic acid probes in solution.

[0493] In a preferred embodiment, the level of mRNA expression is assessed by using branched DNA (bDNA) assay or real-time PCR (qPCR). The use of these methods is described and exemplified in the examples provided herein. In a preferred embodiment, the expression level is determined using a 10 nM siRNA concentration in a species-matched cell line, as provided in Example 2.

[0494] The level of C3 protein expression can be determined using any method known in the art for measuring protein levels, including, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0495] In certain embodiments, the efficacy of the methods of the invention is assessed by a decrease in C3 mRNA or protein levels (eg, in a liver biopsy).

[0496] In some embodiments of the methods of the invention, the iRNA is administered to a subject so that the iRNA is delivered to a specific site within the subject. Inhibition of complement component C3 expression can be assessed using measurements of complement component C3 mRNA or complement component C3 protein levels or changes in samples derived from fluids or tissues (e.g., liver or blood) from the specific site within the subject.

[0497] As used herein, the term detecting or determining the level of an analyte is understood to mean performing a step to determine whether a substance, e.g., protein, RNA, is present. As used herein, a method of detection or determination includes detecting or determining an analyte level that is below the detection level of the method used.

[0498] VII. Prevention and Treatment Methods of the Invention The invention also provides methods for inhibiting expression of complement component C3 using an iRNA of the invention or a composition comprising an iRNA of the invention, thereby preventing or treating complement component C3-associated disorders, such as cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

[0499] In the methods of the present invention, the cell can be contacted with the siRNA in vitro or in vivo, ie, the cell can be within a subject.

[0500] The cell suitable for treatment using the method of the present invention can be any cell that expresses complement component C3 gene, such as liver cell, brain cell, gallbladder cell, heart cell or kidney cell, but is preferably liver cell.The cell suitable for use in the method of the present invention can be mammalian cell, such as primate cell (e.g., human cell or non-human primate cell, including human cell in chimeric non-human animal, for example, monkey cell or chimpanzee cell) or non-primate cell.In some embodiments, the cell is human cell, su...

Claims

1. 1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of complement component C3 in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:

5.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of complement component C3 in a cell, wherein the dsRNA comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region that is complementary to an mRNA encoding complement component C3, and the complementary region comprises at least 15 contiguous nucleotides that differ from either of the antisense nucleotide sequences by no more than 3 nucleotides.

3. 1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of complement component C3 in a cell, wherein the dsRNA comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand is selected from the group consisting of nucleotides 475-497, 487-509, 490-512, 491-513, 705-727, 809-831, 813-835, 1147-1169, 1437-1459, 1439-1461, 1447-1469, 2596-2618, 2634-2656, 3012-3034, 3334-3356, 3611-3633, 3614-3636, 3622-3628, 3639-3640, 3641-3642, 3643-3644, 3645-3646, 3646-3648, 3647-3649, 3650-3651, 3652-3653, 3654-3655, 3656-3657, 3658-3659, 3660-3661, 3662-3663, 3664-3665, 3666-3667, 3668-3669, 3670-3671, 3672-3673, 3674-3675, 3676-3677, 3678-3679, 3680-3681, 3682-3683, 3655, 3809-3831, 3846-3868, 3847-3869, 3920-3942, 4047-4069, 4061-4083, 4156-4178, 4157-4177, 4162-4184, 4178-4200, 4226-4248, 4369-4391, 4392-4414, 4521-4543, 4522-4544, 4523-4545, 5012-5034, and the antisense strand comprises at least 19 contiguous nucleotides of the corresponding nucleotide sequence of SEQ ID NO:

5.

4. 4. The dsRNA agent of claim 3, wherein the sense strand comprises at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from the nucleotide sequence of nucleotides 705-727, 809-831, or 2634-2656 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 19 contiguous nucleotides of the corresponding nucleotide sequence of SEQ ID NO:

5.

5. 5. The dsRNA agent of claim 4, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of nucleotides 2634 to 2656 of SEQ ID NO:1, and the antisense strand comprises at least 19 contiguous nucleotides of the corresponding nucleotide sequence of SEQ ID NO:

5.

6. The antisense strand is AD-565541.2, AD-564742, AD-567304, AD-568978, AD-569164, AD-569272.2, AD-569765.2, AD-564730.2, AD-567315, AD-564745.2, AD-571715.2, AD-570714, AD-571826, AD-572041.2, AD-572039.2, AD-572387, AD-568586.2, AD-566837.2, AD -566444.2, AD-567700.2, AD-567814.2, AD-568003.2, AD-569164.2, AD-569763.2, A 6. The dsRNA agent of any one of claims 1 to 5, comprising at least 15 contiguous nucleotides that differ by 3 or more nucleotides from any of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-565281.2, AD-571539.2, AD-572389.2, AD-567315.2, AD-571752.2, AD-568026.2, AD-571298, AD-572110.2, AD-572062.2, AD-572388.2, AD-572040.2, AD-567713.2, AD-567521.2, AD-567066.2, AD-1181519, AD-569268, or AD-570714.

7. 7. The dsRNA agent of claim 6, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by 3 or more nucleotides from any of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-1181519, AD-569268, or AD-570714.

8. 8. The dsRNA agent of claim 7, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by 3 or more nucleotides from any of the antisense strand nucleotide sequences of the duplex selected from the group consisting of AD-570714.

9. 9. The dsRNA agent of any of claims 1-8, wherein the dsRNA agent comprises at least one modified nucleotide.

10. 10. The dsRNA agent of any of claims 1-9, wherein substantially all of the nucleotides in the sense strand; substantially all of the nucleotides in the antisense strand comprise the modification; or substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand comprise the modification.

11. 11. The dsRNA agent of any of claims 1-10, wherein all nucleotides in the sense strand comprise the modification; all nucleotides in the antisense strand comprise the modification; or all nucleotides in the sense strand and all nucleotides in the antisense strand comprise the modification.

12. at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxy-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, 12. The dsRNA agent of any of claims 9-11, wherein the dsRNA agent is selected from the group consisting of morpholino nucleotides, phosphoramidates, unnatural base-containing nucleotides, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, phosphorothioate-group-containing nucleotides, methylphosphonate-group-containing nucleotides, 5'-phosphate-containing nucleotides, 5'-phosphate-containing nucleotide mimics, thermolabile nucleotides, glycol-modified nucleotides (GNAs), and 2-O-(N-methylacetamido)-modified nucleotides; and combinations thereof.

13. 12. The dsRNA agent of any of claims 9-11, wherein the nucleotide modification is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and glycol; and combinations thereof.

14. 12. The dsRNA agent of any of claims 9-11, wherein at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNAs), and vinyl-phosphonate nucleotides; and combinations thereof.

15. 12. The dsRNA agent of any of claims 9-11, wherein at least one of the nucleotide modifications is a thermolabilizing nucleotide modification.

16. 16. The dsRNA agent of claim 15, wherein the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification; a mismatch with the opposite nucleotide in a duplex; and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an Unlocked Nucleic Acid (UNA), and a Glycerol Nucleic Acid (GNA).

17. 17. The dsRNA agent of any of claims 1-16, wherein the double-stranded region is 19-30 nucleotide pairs in length.

18. 18. The dsRNA agent of claim 17, wherein the double-stranded region is 19 to 25 nucleotide pairs in length.

19. 18. The dsRNA agent of claim 17, wherein the double-stranded region is 19 to 23 nucleotide pairs in length.

20. 18. The dsRNA agent of claim 17, wherein the double-stranded region is 23 to 27 nucleotide pairs in length.

21. 18. The dsRNA agent of claim 17, wherein the double-stranded region is 21 to 23 nucleotide pairs in length.

22. 22. The dsRNA agent of any of claims 1-21, wherein each strand independently is no more than 30 nucleotides in length.

23. 23. The dsRNA agent of any of claims 1-22, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

24. 24. The dsRNA agent of any of claims 1-23, wherein the region of complementarity is at least 17 nucleotides in length.

25. 24. The dsRNA agent of any of claims 1-23, wherein the region of complementarity is 19-23 nucleotides in length.

26. 24. The dsRNA agent of any of claims 1-23, wherein the region of complementarity is 19 nucleotides in length.

27. 27. The dsRNA agent of any of claims 1-26, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

28. 27. The dsRNA agent of any of claims 1-26, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

29. 29. The dsRNA agent of any of claims 1-28, further comprising a ligand.

30. 30. The dsRNA agent of claim 29, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

31. 31. The dsRNA agent of claim 29 or 30, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

32. 32. The dsRNA agent of any of claims 29-31, wherein the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, or trivalent branched linker.

33. The ligand 【Chemical 1】 33. The dsRNA agent of claim 31 or 32, wherein:

34. 34. The dsRNA agent of claim 33, wherein the dsRNA agent is conjugated to a ligand shown in the diagram below. wherein X is O or S.

35. 35. The dsRNA agent of claim 34, wherein X is O.

36. 36. The dsRNA agent of any of claims 1-35, wherein the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

37. 37. The dsRNA agent of claim 36, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand.

38. 38. The dsRNA agent of claim 37, wherein the strand is the antisense strand.

39. 38. The dsRNA agent of claim 37, wherein the strand is the sense strand.

40. 37. The dsRNA agent of claim 36, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand.

41. 41. The dsRNA agent of claim 40, wherein the strand is the antisense strand.

42. 41. The dsRNA agent of claim 40, wherein the strand is the sense strand.

43. 37. The dsRNA agent of claim 36, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand.

44. 44. The dsRNA agent of claim 43, wherein the strand is the antisense strand.

45. 45. The dsRNA agent of any one of claims 1 to 44, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

46. 46. ​​A cell comprising the dsRNA agent of any of claims 1-45.

47. 46. ​​A pharmaceutical composition for inhibiting expression of a gene encoding complement component C3, comprising the dsRNA agent of any of claims 1-45.

48. 48. The pharmaceutical composition of claim 47, wherein the dsRNA agent is in an unbuffered solution.

49. 49. The pharmaceutical composition of claim 48, wherein the unbuffered solution is saline or water.

50. 48. The pharmaceutical composition of claim 47, wherein the dsRNA agent is in a buffer solution.

51. 51. The pharmaceutical composition of claim 50, wherein the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

52. 52. The pharmaceutical composition of claim 51, wherein the buffer is phosphate buffered saline (PBS).

53. 52. A method of inhibiting expression of the complement component C3 gene in a cell, comprising contacting the cell with the dsRNA agent of any of claims 1-45 or the pharmaceutical composition of any of claims 47-52, thereby inhibiting expression of the complement component C3 gene in the cell.

54. 54. The method of claim 53, wherein the cell is in a subject.

55. 55. The method of claim 54, wherein the subject is a human.

56. 56. The method of claim 55, wherein the subject has a complement component C3-associated disorder.

57. 57. The method of claim 56, wherein the complement component C3-related disorder is selected from the group consisting of cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, e.g., pemphigus vulgaris (PV) and pemphigus foliaceus (PF), and C3 glomerulopathy.

58. 58. The method of any of claims 53-57, wherein contacting the cell with the dsRNA agent inhibits expression of complement component C3 by at least 50%, 60%, 70%, 80%, 90%, or 95%.

59. 59. The method of any of claims 54-58, wherein inhibiting expression of complement component C3 reduces complement component C3 protein levels in the subject's serum by at least 50%, 60%, 70%, 80%, 90%, or 95%.

60. A method of treating a subject having a disorder that would benefit from reduced expression of complement component C3, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any of claims 1-45 or the pharmaceutical composition of any of claims 47-52, thereby treating the subject having a disorder that would benefit from reduced expression of complement component C3.

61. 52. A method of preventing at least one symptom in a subject having a disorder that would benefit from reduced expression of complement component C3, comprising administering to the subject a prophylactically effective amount of the dsRNA agent of any of claims 1-45 or the pharmaceutical composition of any of claims 47-52, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduced expression of complement component C3.

62. 62. The method of claim 60 or 61, wherein the disorder is a complement component C3-associated disorder.

63. 63. The method of claim 62, wherein the complement component C3-related disorder is selected from the group consisting of cold agglutinin disease (CAD), warm autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria (PNH), lupus nephritis (LN), pemphigoid, pemphigus, pemphigus vulgaris (PV) and pemphigus foliaceus (PF) and C3 glomerulopathy.

64. 63. The method of claim 62, wherein the complement component C3-associated disorder is cold agglutinin disease (CAD).

65. 63. The method of claim 62, wherein the subject is a human.

66. 62. The method of claim 60 or 61, wherein administration of the agent to the subject causes a decrease in hemolysis and / or a decrease in C3 protein accumulation.

67. 67. The method of any of claims 60-66, wherein the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

68. 68. The method of any of claims 60-67, wherein the dsRNA agent is administered to the subject subcutaneously.

69. 69. The method of any of claims 60-68, further comprising determining the level of complement component C3 in a sample from the subject.

70. 70. The method of claim 69, wherein the level of complement component C3 in the subject sample is the complement component C3 protein level in a blood or serum sample.

71. 71. The method of any of claims 60-70, further comprising administering to the subject an additional therapeutic agent for the treatment of hemolysis.

72. 53. A kit comprising the dsRNA agent of any of claims 1-45 or the pharmaceutical composition of any of claims 47-52.

73. 53. A vial comprising the dsRNA agent of any of claims 1-45 or the pharmaceutical composition of any of claims 47-52.

74. 53. A syringe comprising the dsRNA agent of any of claims 1-45 or the pharmaceutical composition of any of claims 47-52.

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